Retentate chromatography and protein chip arrays with applications in biology and medicine

ABSTRACT

This invention provides methods of retentate chromatography for resolving analytes in a sample. The methods involve adsorbing the analytes to a substrate under a plurality of different selectivity conditions, and detecting the analytes retained on the substrate by desorption spectrometry. The methods are useful in biology and medicine, including clinical diagnostics and drug discovery.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of the priority dates of nowabandoned application No. 60/054,333 filed Jun. 20, 1997 and nowabandoned application No. 60/067,484 filed Dec. 1, 1997, the contents ofwhich are incorporated herein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

This invention relates to the field of separation science and analyticalbiochemistry.

The methods of this invention have applications in biology and medicine,including analysis of gene function, differential gene expression,protein discovery, cellular and clinical diagnostics and drug screening.

Cell function, both normal and pathologic, depends, in part, on thegenes expressed by the cell (i.e., gene function). Gene expression hasboth qualitative and quantitative aspects. That is, cells may differboth in terms of the particular genes expressed and in terms of relativelevel of expression of the same gene. Differential gene expression canbe manifested, for example, by differences in the expression of proteinsencoded by the gene, or in post-translational modifications of expressedproteins. For example, proteins can be decorated with carbohydrates orphosphate groups, or they can be processed through peptide cleavage.Thus, at the biochemical level, a cell represents a complex mixture oforganic biomolecules.

One goal of functional genomics (“proteomics”) is the identification andcharacterization of organic biomolecules that are differentiallyexpressed between cell types. By comparing expression one can identifymolecules that may be responsible for a particular pathologic activityof a cell. For example, identifying a protein that is expressed incancer cells but not in normal cells is useful for diagnosis and,ultimately, for drug discovery and treatment of the pathology. Uponcompletion of the Human Genome Project, all the human genes will havebeen cloned, sequenced and organized in databases. In this “post-genome”world, the ability to identify differentially expressed proteins willlead, in turn, to the identification of the genes that encode them.Thus, the power of genetics can be brought to bear on problems of cellfunction.

Differential chemical analyses of gene expression and function requiretools that can resolve the complex mixture of molecules in a cell,quantify them and identify them, even when present in trace amounts.However, the current tools of analytical chemistry for this purpose arelimited in each of these areas. One popular biomolecular separationmethod is gel electrophoresis. Frequently, a first separation ofproteins by isoelectric focusing in a gel is coupled with a secondseparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The result is a map that resolves proteins according to thedimensions of isoelectric point (net charge) and size (i.e., mass).However useful, this method is limited in several ways. First, themethod provides information only about two characteristics of abiomolecule—mass and isoelectric point (“pI”). Second, the resolutionpower in each of the dimensions is limited by the resolving power of thegel. For example, molecules whose mass differ by less than about 5% orless than about 0.5 pI are often difficult to resolve. Third, gels havelimited loading capacity, and thus sensitivity; one may not be able todetect biomolecules that are expressed in small quantities. Fourth,small proteins and peptides with a molecular mass below about 10-20 kDaare not observed.

Other analytical methods may overcome one or more of these limitations,but they are difficult to combine efficiently. For example, analyticalchromatography can separate biomolecules based on a variety ofanalyte/adsorbent interactions, but multi-dimensional analysis isdifficult and time consuming. Furthermore, the methods are limited insensitivity.

Clinical diagnostics requires the ability to specifically detect knownmarkers of disease. However, the development of such diagnostics ishampered by the time necessary to prepare reagents that specificallybind to markers, or that can discriminate the marker in a complexmixture.

Drug discovery requires the ability to rapidly screen agents thatmodulate ligand/receptor interactions. Often the rate-limiting step insuch screens is the ability to detect the ligand/receptor interaction.Thus, rapid and specific methods for identifying binding events would bean advance in the art.

Until now, the process from identifying a potential marker or member ofa ligand/receptor pair to producing an agent that specifically binds themarker or member has been difficult. In one method, normal and diseasedtissue are compared to identify mRNA species or expressed sequence tags(“ESTs”) that are elevated or decreased in the diseased tissue. Thesespecies are isolated and the polypeptides they encode are producedthrough routine methods of recombinant DNA. Then, the polypeptides areisolated and used as immunogens to raise antibodies specific for themarker. The antibodies can be used in, for example, ELISA assays todetermine the amount of the marker in a patient sample.

This process is long and tedious. It can take nine months to a year toproduce such antibodies, with much of the time being spent on developingprotocols to isolate a sufficient quantity of the polypeptide forimmunization. Furthermore, the method relies on the hope thatdifferences in RNA expression are expressed as differences in proteinexpression. However, this assumption is not always reliable. Therefore,methods in which differentially expressed proteins are detected directlyand in which specific ligands could be generated in significantlyshorter time would be of great benefit to the field.

Thus, tools for resolving complex mixtures of organic biomolecules,identifying individual biomolecules in the mixture and identifyingspecific molecular recognition events involving one or more targetanalytes are desirable for analytical biochemistry, biology andmedicine.

SUMMARY OF THE INVENTION

This invention provides devices and methods for retentatechromatography. Retentate chromatography is a combinatorial method toprovide high information resolution of analytes in complex mixturesthrough the use of multi-dimensional separation methods. It provides aunified analyte detection and functional analysis capability for biologyand medicine that is characterized by a single, integrated operatingsystem for the direct detection of analyte expression patternsassociated with gene function, protein function, cell function, and thefunction of whole organisms. In one aspect, this invention provides aunified operating system for the discovery or diagnosis of genefunction, protein function, or the function of entire macromolecularassemblies, cells, and whole organisms.

More particularly, analytes can be resolved in a variety oftwo-dimensional formats, thereby providing multi-dimensionalinformation. Analytes are first separated in at least two differentfirst dimensions based on their ability to be adsorbed to a stationaryphase under at least two different selectivity conditions, such asanionic/cationic potential, hydrophobicity/hydrophilicity, or specificbiomolecular recognition. Then the analytes are separated in a seconddimension based on mass by desorption spectrometry (e.g., laserdesorption mass spectrometry), which further provides detection of theseparated analytes. The nature of the adsorbent to which the analytesadsorb provides physico-chemical information about the analyte.

Thus, this invention provides a molecular discovery and diagnosticdevice that is characterized by the inclusion of both parallel andmultiplex analyte processing capabilities. Because analytes are directlydetected, the invention enables the simultaneous transmission of two ormore independent target analyte signals from the same “circuit” (i.e.,addressable “chip” location) during a single unit operation.

Retentate chromatography is distinct from conventional chromatography inseveral ways. First, in retentate chromatography, analytes which areretained on the adsorbent are detected. In conventional chromatographicmethods analytes are eluted off of the adsorbent prior to detection.There is no routine or convenient means for detecting analyte which isnot eluted off the adsorbent in conventional chromatography. Thus,retentate chromatography provides direct information about chemical orstructural characteristics of the retained analytes. Second, thecoupling of adsorption chromatography with detection by desorptionspectrometry provides extraordinary sensitivity, in the femtomolarrange, and unusually fine resolution. Third, in part because it allowsdirect detection of analytes, retentate chromatography provides theability to rapidly analyze retentates with a variety of differentselectivity conditions, thus providing rapid, multi-dimensionalcharacterization of analytes in a sample. Fourth, adsorbents can beattached to a substrate in an array of pre-determined, addressablelocations. This allows parallel processing of analytes exposed todifferent adsorbent sites (i.e., “affinity sites” or “spots”) on thearray under different elution conditions.

Retentate chromatography has many uses in biology and medicine. Theseuses include combinatorial biochemical separation and purification ofanalytes, the study of differential gene expression and molecularrecognition events, diagnostics and drug discovery.

One basic use of retentate chromatography as an analytical tool involvesexposing a sample to a combinatorial assortment of differentadsorbent/eluant combinations and detecting the behavior of the analyteunder the different conditions. This both purifies the analyte andidentifies conditions useful for detecting the analyte in a sample.Substrates having adsorbents identified in this way can be used asspecific detectors of the analyte or analytes. In a progressiveextraction method, a sample is exposed to a first adsorbent/eluantcombination and the wash, depleted of analytes that are adsorbed by thefirst adsorbent, is exposed to a second adsorbent to deplete it of otheranalytes. Selectivity conditions identified to retain analytes also canbe used in preparative purification procedures in which an impure samplecontaining an analyte is exposed, sequentially, to adsorbents thatretain it, impurities are removed, and the retained analyte is collectedfrom the adsorbent for a subsequent round.

One aspect of the invention is that each class or type of molecularrecognition event (e.g., target adsorbent-target analyte interaction),characterized by a particular selectivity condition at an addressablelocation within the array, is detected directly while the associatedmolecules are still localized (i.e., “retained”) at the addressablelocation. That is, selection and detection, by direct means, does notrequire elution, recovery, amplification, or labeling of the targetanalyte.

Another aspect of the present invention is that the detection of one ormore desirable molecular recognition events, at one or more locationswithin the addressable array, does not require removal or consumption ofmore than a small fraction of the total adsorbent-analyte. Thus, theunused portion can be interrogated further after one or more “secondaryprocessing” events conducted directly in situ (i.e., within the boundaryof the addressable location) for the purpose of structure and functionelucidation, including further assembly or disassembly, modification, oramplification (directly or indirectly).

Adsorbents with improved specificity for an analyte can be developed byan iterative process, referred to as “progressive resolution,” in whichadsorbents or eluants proven to retain an analyte are tested withadditional variables to identity combinations with better bindingcharacteristics. Another method allows the rapid creation of substrateswith antibody adsorbents specific for an analyte. The method involvesdocking the analyte to an adsorbent, and screening phage displaylibraries for phage that bind the analyte.

Retentate chromatography has uses in molecular and cellular biology, aswell. Analytes that are differentially present in two samples (e.g.,differentially expressed proteins in two cell extracts) can beidentified by exposing the samples to a variety of adsorbent/eluantcombinations for analysis by desorption spectrometry, thereby making useof the high information resolving power of the system that otherseparation and detections systems cannot match. Unknown target proteinscan be identified by determining physicochemical characteristics,including molecular mass, based on the chemical characteristics of theadsorbent/eluant combination, and this information can be used to screendatabases for proteins having similar profiles.

The methods in separation biochemistry and the adsorbents produced fromthese methods, are useful in diagnostics. More particularly, adsorbents,either chemical or biospecific, can be developed to detect importantdiagnostic markers. In certain embodiments, a substrate can have anarray of adsorbent spots selected for a combination of markersdiagnostic for a disease or syndrome.

Retentate chromatography also is useful in drug discovery. One member ofa receptor/ligand pair is docked to an adsorbent, and its ability tobind the binding partner is tested in the presence of the agent. Becauseof the rapidity with which adsorption can be tested, combinatoriallibraries of agents can be easily tested for their ability to modulatethe interaction.

In one aspect this invention provides a method for high informationresolution of at least one analyte in a sample. The method is acombinatorial separation method that includes separation and detectionof multiple analytes in parallel. The method comprises the steps of a)exposing the analyte to at least two different selectivity conditions,each selectivity condition defined by the combination of an adsorbentand an eluant, to allow retention of the analyte by the adsorbent; andb) detecting retained analyte under the different selectivity conditionsby desorption spectrometry. Detection of retained analyte under thedifferent selectivity conditions provides a high information resolutionof the analyte.

In one embodiment each different selectivity condition is defined at adifferent predetermined, addressable location for parallel processing.In another embodiment, the method comprises the steps of i) exposing theanalyte to a first selectivity condition at a defined location to allowretention of the analyte by the adsorbent; ii) detecting retainedanalyte under the first selectivity condition by desorptionspectrometry; iii) washing the adsorbent under a second, differentselectivity condition at the defined location to allow retention of theanalyte to the adsorbent, and iv) detecting retained analyte under thesecond selectivity condition by desorption spectrometry.

In another embodiment the analyte is an organic biomolecule, amultimeric molecular complex or macromolecular assembly. In anotherembodiment the organic biomolecule is an enzyme, an immunoglobulin, acell surface receptor or an intracellular receptor.

In another embodiment the adsorbent comprises an anion, a cation, ahydrophobic interaction adsorbent, a polypeptide, a nucleic acid, acarbohydrate, a lectin, a dye, a reducing agent, a hydrocarbon or acombination thereof. In another embodiment the adsorbent is attached toa substrate comprising glass, ceramic, a magnetic material, an organicpolymer, a conducting polymer, a native biopolymer, a metal or a metalcoated with an organic polymer. In another embodiment the adsorbent isin the form of a microemulsion, a latex, a layer or a bead. In anotherembodiment the locations on the substrate are arranged in a line or anorthogonal array. In another embodiment the adsorbents are located on asubstrate at different locations before the analytes are exposed to theselectivity conditions. In another embodiment the adsorbents are locatedon a substrate at different locations after the analytes are exposed tothe selectivity conditions. In another embodiment the differentselectivity conditions comprise different binding conditions ordifferent elution conditions.

In another embodiment the step of detecting comprises detecting the massof the analyte by laser desorption mass spectrometry.

In another embodiment the selectivity conditions are selected tooptimize retention of analyte by an adsorbent. In another embodiment theat least one analyte is more than one analyte. In another embodiment theplurality of selectivity conditions are defined by at differentadsorbents and the same eluant.

Another embodiment further comprises the step of providing a substratecomprising adsorbents at addressable locations, each adsorbent being anadsorbent from a selectivity condition identified to retain the analyte.In another embodiment the elution conditions differ according to pH,buffering capacity, ionic strength, a water structure characteristic,detergent type, detergent strength, hydrophobicity or dielectricconstant. In another embodiment the plurality of selectivity conditionsare defined by the same eluant.

In another embodiment this invention provides a method for sequentialextraction of analytes from a sample. This is a combinatorial, serialseparation and purification development method for multiple analytes inparallel. The method comprises the steps of a) exposing a samplecomprising analytes to a first selectivity condition to allow retentionof analytes by a first adsorbent and to create un-retained sample; b)collecting the un-retained sample comprising analytes, exposing theun-retained sample to a second selectivity condition to allow retentionof analytes by a second adsorbent and to create un-retained sample; andc) detecting retained analyte under the different selectivity conditionsby desorption spectrometry.

In another aspect this invention provides a substrate for desorptionspectrometry comprising an adsorbent whose binding characteristics varyin a gradient along one or more linear axes.

In another aspect this invention provides a method for progressivelyidentifying a selectivity condition with improved resolution for ananalyte in a sample. The method comprises the steps of: (a) identify aselectivity condition that retains an analyte in a sample by (i)exposing a sample to a set of selectivity conditions, each selectivitycondition defined by at least one binding characteristic and at leastone elution characteristic; (ii) detecting analyte retained under eachselectivity condition by desorption spectrometry; and (iii) identifyinga selectivity condition that retains the analyte; and (b) identifying aselectivity condition with improved resolution for the analyte by: (i)selecting at least one binding characteristic or elution characteristicfrom the identified selectivity condition and adding it to a selectivitycharacteristic constant set; (ii) exposing the sample to a modified setof selectivity conditions wherein each selectivity condition in themodified set comprises (1) the selectivity characteristics in theconstant set and (2) a binding characteristic or elution characteristicthat is not in the constant set; and (iii) identifying a selectivitycondition from the modified set by desorption spectrometry that retainsthe analyte with improved resolution compared with a prior identifiedselectivity condition. One embodiment comprises the step of repeatingstep (b) at least once. Another embodiment comprises repeating steps (b)until a selectivity condition is identified that retains only the targetanalyte from the sample.

In another aspect this invention provides a substrate for desorptionspectrometry comprising an adsorbent from a selectivity conditionsidentified to resolve an analyte by the method of progressiveresolution. In one embodiment the substrate comes in the form of a kitfurther comprising an eluant from the selectivity condition orinstructions on using the eluant in combination with the adsorbent.

In another aspect this invention provides a method for preparativepurification an analyte from an impure sample. The method comprises thesteps of a) exposing the sample to a substrate under a plurality ofdifferent selectivity conditions; detecting retained analyte under thedifferent selectivity conditions by desorption spectrometry; andidentifying selectivity conditions under which the analyte is retained;b) purifying the analyte by repeating, for a plurality of differentidentified selectivity conditions, a sequence of steps comprising i)exposing the sample to an adsorbent under the identified selectivitycondition to allow retention of the analyte by the adsorbent; ii)separating the analyte from an impurity that is not retained by thesubstrate; and iii) collecting the analyte from the adsorbent.

In another aspect this invention provides a method for preparing asubstrate for detecting at least one analyte in a sample. This method isa combinatorial method for the design and identification ofanalyte-specific adsorbents. It is useful in detecting target analytes.The method comprises the steps of a) exposing the sample to at least twodifferent selectivity conditions, each selectivity condition defined bythe combination of an adsorbent and an eluant, to allow retention of theanalyte by the adsorbent; b) identifying by desorption spectrometry atleast one selectivity condition under which the analyte is retained; andc) preparing a substrate comprising at least one adsorbent of anidentified selectivity condition. In one embodiment, the step ofidentifying comprises identifying at least one selectivity conditionunder which a plurality of analytes are retained. In another embodimentthe step of preparing comprises preparing a substrate comprising aplurality of adsorbents that retain the analyte under an elutioncondition as a multiplex adsorbent.

In another aspect this invention provides a method of diagnosing in asubject a disease characterized by at least one diagnostic marker. Thisis a combinatorial method for simultaneous detection of multiplediagnostic markers. The method comprises the steps of a) providing asubstrate for use in desorption spectrometry that comprises at least oneaddressable location, each addressable location comprising an adsorbentthat resolves at least one of the diagnostic markers under an elutioncondition; b) exposing the substrate to a biological sample from thesubject under the elution condition to allow retention of the diagnosticmarker; and c) detecting retained diagnostic marker by desorptionspectrometry. Detecting retained diagnostic marker provides a diagnosisof the disease.

In another aspect this invention provides a kit for detecting an analytein a sample comprising (1) a substrate for use in desorptionspectrometry that comprises at least one addressable location, eachaddressable location comprising an adsorbent that resolves an analyteunder a selectivity condition comprising the adsorbent and an eluant,and (2) the eluant or instructions for exposing the sample to theselectivity condition. In one embodiment the kit is characterized by aplurality of diagnostic markers and the substrate comprises a pluralityof addressable locations, each addressable location comprising anadsorbent that resolves at least one of the diagnostic markers.

In another aspect this invention provides a substrate for desorptionspectrometry comprising at least one adsorbent in at least oneaddressable location wherein the at least one adsorbent resolves aplurality of diagnostic markers for a pathological condition from apatient sample.

In another aspect this invention provides a method for selectingidentity candidates for an analyte protein. This method is acombinatorial method for protein identification based on at least twophysico-chemical properties. The method comprises the steps of a)determining a value set specifying match parameters for at least a firstand second physico-chemical characteristic of a protein analyte in asample by i) exposing the analyte to a plurality of differentselectivity conditions, wherein adsorption of the protein analyte to thesubstrate is mediated by a basis of attraction that identifies aphysico-chemical characteristic of the protein analyte; and ii)detecting retained analyte under the different selectivity conditions bydesorption spectrometry; and b) performing, in a programmable digitalcomputer, the steps of i) accessing a database comprising, for eachmember of a set of reference polypeptides, a value set specifying atleast a first and second physico-chemical characteristic of thereference polypeptides; ii) inputting the value set specifying thephysico-chemical characteristics of the protein analyte; iii) sortingfrom the database, reference polypeptides having value sets within thematch parameters. The sorted reference polypeptides provide identitycandidates for the protein analyte. Unsorted references polypeptides arethose excluded as identity candidates.

In another aspect this invention provides a method for sequentiallyretaining analytes. This method is a multimeric macromolecular orsupramolecular assembly monitoring method. It is useful as a method fordrug discovery by molecular recognition interference. The methodcomprises the steps of a) exposing a first sample to a primary adsorbentand to an eluant to allow retention of a first analyte by the adsorbent,and detecting the adsorbed analyte by desorption spectrometry, wherebythe retained first analyte becomes a secondary adsorbent; b) exposing asecond sample to the secondary adsorbent and to an eluant to allowretention of a second analyte by the secondary adsorbent, and detectingthe adsorbed second analyte by desorption spectrometry, whereby theretained second analyte becomes a tertiary adsorbent.

In another aspect this invention provides a method of detecting anenzyme in a sample. The method comprises the steps of: a) providing asolid phase comprising an adsorbent and an enzyme substrate bound to theadsorbent, wherein the activity of the enzyme on the enzyme substrateproduces a product having a characteristic molecular mass; b) exposingthe substrate to the sample; and c) detecting the product by desorptionspectrometry. Detecting the product provides a detection of the enzyme.

In another aspect this invention provides a method for determiningwhether an analyte is differentially present (e.g., differentiallyexpressed) in a first and second biological sample. The method is usefulfor combinatorial method for differential gene expression monitoring bydifferential protein display. The method comprises the steps of a)determining a first retention map for the analyte in the first samplefor at least one selectivity condition; b) determining a secondretention map for the analyte in the second sample for the sameselectivity condition; and c) detecting a difference between the firstand the second retention maps. A difference in the retention mapsprovides a determination that the analyte is differentially present infirst and second samples.

In one embodiment the method is for determining whether a protein isdifferentially expressed between two different cells, and the first andsecond samples comprise the cells or material from the cells. In anotherembodiment the method if for determining whether an agent alters theexpression of a protein in a biological sample further comprising thestep of administering the agent to a first biological sample but not toa second biological sample. In another embodiment the first biologicalsample derives from a healthy subject and the second biological sampleis from a subject suffering from a pathological condition. The samplecan be selected from, for example, blood, urine, serum and tissue.Analytes that are found to be increased in samples from pathologicalsubjects are candidate diagnostic markers. Generally, confirmation of adianostic marker involves detection of the marker in many subjects.

In another aspect this invention provides a method for identifying aligand for a receptor. The method comprises the steps of: a) providing asubstrate comprising an adsorbent wherein the receptor is bound to theadsorbent; b) exposing the bound receptor to a sample containing theligand under conditions to allow binding between the receptor and theligand; and c) detecting bound ligand by desorption spectrometry.

In another aspect this invention provides a screening method fordetermining whether an agent modulates binding between a target analyteand an adsorbent. This is a combinatorial method for drug discovery. Themethod comprises the steps of a) providing a substrate comprising anadsorbent to which the target analyte binds under an elution condition;b) exposing the substrate to the target analyte and to the agent underthe elution condition to allow binding between the target analyte andthe adsorbent; c) detecting an amount of binding between the targetanalyte and the adsorbent by desorption spectrometry; and d) determiningwhether the measured amount is different than a control amount ofbinding when the substrate is exposed to the target analyte under theelution condition without the agent. A difference between the measuredamount and the control amount indicates that the agent modulatesbinding.

In one aspect, this invention provides a method of detecting a geneticpackage containing a polynucleotide that encodes a polypeptide agentthat specifically binds to a target adsorbent. This is, in one aspect, acombinatorial method for selecting analyte-specific phage from a displaylibrary, including the use of target proteins isolated by retentatemapping or target proteins generated in situ by in vitro transcriptionand translation. The method comprises the steps of: a) providing asubstrate comprising a target adsorbent; b) providing a display librarythat comprises a plurality of different genetic packages, each differentgenetic package comprising a polynucleotide that comprises a nucleotidesequence that encodes a polypeptide agent, and each different geneticpackage having a surface on which the encoded polypeptide agent isdisplayed; c) exposing the substrate to the display library underelution conditions to allow specific binding between a polypeptide agentand the target adsorbent, whereby a genetic package comprising thepolypeptide agent is retained on the substrate; and d) detecting agenetic package retained on the substrate by desorption spectrometry.

In one embodiment of this method, the display library is a phage displaylibrary. In another embodiment the phage is M13. In another embodimentthe polypeptide is a single chain antibody. In another embodiment thetarget analyte is a polypeptide analyte that is differentially expressedbetween cells of different phenotypes. In another embodiment thesubstrate comprises a cell or cell membrane.

In one embodiment, the step of providing the substrate comprising thetarget adsorbent comprises the steps of: i) providing a substratecomprising an adsorbent, wherein the adsorbent retains a target analyteunder an elution condition; and ii) exposing the adsorbent to the targetanalyte under the elution condition to allow retention of the targetanalyte by the adsorbent, whereby the target analyte becomes the targetadsorbent. In one embodiment, the target analyte is a target polypeptideand the step of ii) exposing the adsorbent comprises the step ofproducing the target polypeptide in situ on the adsorbent by in vitrotranslation of a polynucleotide encoding the target polypeptide, and canfurther comprise amplifying the polynucleotide sequence in situ on thesubstrate.

In another embodiment the substrate comprises (1) an adsorbent thatbinds an anchoring polypeptide and (2) at least one target geneticpackage having a surface displaying the anchoring polypeptide and atarget adsorbent polypeptide, the target genetic package comprising apolynucleotide that comprises a nucleotide sequence that encodes thetarget adsorbent, wherein the target genetic package is bound to theadsorbent through the anchoring polypeptide.

In another embodiment the method further comprises any of the followingsteps: sequencing the nucleotide sequence that encodes the polypeptideagent; isolating the retained genetic package or producing thepolypeptide agent.

In another aspect this invention provides a substrate for desorptionspectrometry comprising an adsorbent that binds an anchoring polypeptidedisplayed on a surface of a genetic package, wherein the surface of thegenetic package further displays a target polypeptide and wherein thegenetic package comprises a polynucleotide comprising a nucleotidesequence that encodes the target polypeptide.

In another aspect this invention provides a method for detectingtranslation of a polynucleotide. The method comprises the steps of: a)providing a substrate comprising an adsorbent for use in desorptionspectrometry; b) contacting the substrate with the polynucleotideencoding a polypeptide and with agents for in vitro translation of thepolynucleotide, whereby the polypeptide is produced; c) exposing thesubstrate to an eluant to allow retention of the polypeptide by theadsorbent; and d) detecting retained polypeptide by desorptionspectrometry. Detection of the polypeptide provides detection oftranslation of the polynucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a substrate containing a plurality of adsorbent spots inthe form of a strip. The strip contains six different sets adsorbentsclassified according to a basis of attraction (hydrophobic, ionic,coordinate covalent and mixed function). The strip contains severalspots for each type of adsorbent, allowing interrogation of the spots atdifferent times with different eluants, or for archiving and subsequentanalysis.

FIG. 2 depicts an orthogonal array of adsorbents (surface interactionpotentials) in predetermined, addressable locations. The array also cantake the form of a plate. The array includes various adsorbents. Uponexposure to the analyte, each strip can be washed by a variety ofeluants (selectivity threshold modifiers). Analysis of retention underdifferent selectivity conditions results in retention map or recognitionprofile.

FIG. 3 is a representation of the quantitative analysis of analytes bydesorption of analyte from given locations on the array and quantitativedetection of the desorbed analyte by laser desorption mass spectrometry.

FIG. 4A illustrates an example of a computer system used to executesoftware that can be used to analyze data generated by the presentinvention. FIG. 4A shows a computer system 1 which includes a monitor 3,screen 5, cabinet 7, keyboard 9, and mouse 11. Mouse 11 may have one ormore buttons such as mouse buttons 13. Cabinet 7 houses a CD-ROM drive15 and a hard drive (not shown) that may be utilized to store andretrieve computer programs including code incorporating the presentinvention. Although a CD-ROM 17 is shown as the computer readablestorage medium, other computer readable storage media including floppydisks, DRAM, hard drives, flash memory, tape, and the like may beutilized. Cabinet 7 also houses familiar computer components (not shown)such as a processor, memory, and the like.

FIG. 4B shows a system block diagram of computer system 1 used toexecute software that can be used to analyze data generated by thepresent invention. As in FIG. 4A, computer system 1 includes monitor 3and keyboard 9. Computer system 1 further includes subsystems such as acentral processor 102, system memory 104, I/O controller 106, displayadapter 108, removable disk 112, fixed disk 116, network interface 118,and speaker 120. Removable disk 112 is representative of removablecomputer readable media like floppies, tape, CD-ROM, removable harddrive, flash memory, and the like. Fixed disk 116 is representative ofan internal hard drive, DRAM, or the like. Other computer systemssuitable for use with the present invention may include additional orfewer subsystems. For example, another computer system could includemore than one processor 102 (i.e., a multi-processor system) or memorycache.

FIGS. 5A-5F show retention maps for lysozyme under selectivityconditions including six different adsorbents and several differenteluants.

FIGS. 6A-6B show the resolution at low and high molecular mass ofanalytes in human serum by an immobilized metal adsorbent.

FIGS. 7A-7B show the resolution at low and high molecular mass ofanalytes in human serum by a variety of adsorbents using the sameeluant.

FIGS. 8A-8B show the resolution at low and high molecular mass ofanalytes in preterm infant urine by a variety of adsorbents using wateras the eluant.

FIG. 9 shows resolution of analytes in preterm infant urine using ahydrophobic phenyl adsorbent and three different eluants, resulting inthe discovery of selective retention of one of the analytes (*) by theTWEEN™ (polyoxyethylenesorbitan) wash condition.

FIGS. 10A-10D show the resolution of analytes in cell culture medium oftwo different breast cancer cell lines.

FIG. 11 shows a composite retention map of preterm infant urine exposedto selectivity conditions defined by six different adsorbents and threedifferent eluants.

FIG. 12 shows a two-dimensional polyacrylamide gel (pI and apparentmolecular mass) of preterm infant urine.

FIG. 13 shows a method of panning with phage display libraries for aphage having a surface protein that specifically binds to a targetanalyte. The substrate depicted at the top shows that even a fewspecifically bound phage can be detected by desorption spectrometrythrough the detection of the many coat proteins that phage contains. Atthe bottom, a substrate with several adsorbent spots is developed sothat the target analyte is specifically bound. Phage are exposed to thespots. Bound phage are detected by desorption spectrometry. Phage boundto another spot can be isolated and grown.

FIG. 14 shows how a ligand agent, in this case a single chain antibody,identified by a panning method can be used as an adsorbent to dock atarget protein for use in protein-protein interaction studies. A targetis purified in situ (spot 2) and used to pan a phage display library(spot 4). A single chain antibody is isolated and attached to asubstrate (spot 6) as an adsorbent. The target is then adsorbed to thesingle chain antibody. The target is now docked for the study ofprotein-protein interactions (spot 8).

FIG. 15 shows a method for screening drug candidates for the ability tointerfere with protein binding to a ligand, in this case a single-chainantibody. A single chain antibody specific for a target protein isdocked to a spot on a substrate through, for example, an anti-phageantibody which, itself, can be docked through protein A or protein G.The single chain antibody is exposed to the target protein and to drugcandidates. The ability of the drug to bind to the analyte protein andto interfere with ligand binding to analyte is monitored by desorptionspectrometry.

FIG. 16 shows a method for screening drug candidates for the ability tointerfere with protein binding to a ligand. The method is similar tothat depicted in the previous figure, except one monitors the ability ofthe drug to interfere with analyte binding by binding, itself, to theligand by desorption spectrometry.

FIG. 17 shows a method for screening drug candidates for the ability tointerfere with target protein (Target protein I) binding to a secondaryligand (Target Protein II). As in the previous two figures, the targetis docked to the substrate becoming, itself, an adsorbent for theligand. In this case, the analyte is docked through a single chainantibody. The target is then exposed to the ligand and to the drugcandidates. The ability of the drug to interfere with binding betweenthe analyte and the ligand (by, e.g., binding to the target analyte) ismonitored by desorption spectrometry.

FIG. 18 depicts a flow chart beginning with the identification ofdifferentially expressed mRNA or polypeptides and ending with thecreation of a diagnostic platform for specifically binding thepolypeptide for detection by desorption spectrometry.

FIGS. 19A-19D show a retention map of Hemophilus lysate on an adsorbentarray. FIG. 19A: anionic adsorbent; FIG. 19B: Normal phase adsorbent;FIG. 19C: Ni(II) adsorbent; FIG. 19D: Hydrophobic adsorbent.

FIGS. 20A-20C show progressive resolution of an analyte in Hemophiluslysate. The adsorbent in each case was an anionic adsorbent. FIG. 20A:In a first step, after exposure to the sample, the spot was washed with150 μl of 20 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0. In asecond step, the adsorbent and sodium phosphate characteristic of theeluant were added to a constant set of characteristics. A new elutioncharacteristic was added. FIG. 20B: In addition to 20 mM sodiumphosphate, pH 7.0, the spot was washed with 0.05% Triton X100 and 0.15 MNaCl (150 μl, total). FIG. 20C: In addition to 20 mM sodium phosphate,pH 7.0, the spot was washed with 100 mM imidazole, 0.15 M NaCl (150 μltotal).

FIGS. 21A-21D show the results of a comparison between components innormal human serum and diseased serum. FIG. 21A: Retentate map of normalserum on an adsorbent array Cu(II) site. FIG. 21B: Retentate map ofdisease serum on an adsorbent array Cu(II) site. FIG. 21C: Retainedanalytes of both serum samples are combined in an overlay fashion. Tosimplify the presentation, each peak of retained analyte is converted toa bar, the dashed bars represent analytes retained from a normal serum,and the solid bars represent analytes retained from a disease serum.FIG. 21D: To differentiate more clearly the difference between the twosamples, a comparison plot is generated, where the ratio of the retainedanalytes from the samples are calculated and displayed. The two analytesmarked with “*” show significant increases in the disease serum (5 to 10fold increases).

FIGS. 22A-22D show a comparison of retentate maps for control, diseasedand drug-treated mouse urine on a Cu(II) adsorbent, and quantitation ofamount of a marker in diseased and drug-treated urine.

FIGS. 23A-23D show retentate maps of analytes in urine from four humancancer patients shown in “gel view” format. Difference maps betweenpatients 1, 2 and 3 show two common analytes that are present inincreased amounts in these patients.

FIGS. 24A-24E show detection of M13 phage by laser desorption massspectrometry through the detection of the gene VIII coat protein. Thedilutions of the original 10¹² phage per ml range from 1:10 to1:100,000,000.

FIGS. 25A-25B show the capture of M13 by desorption spectrometry usinganti-M13 antibody as an adsorbent. FIG. 22A shows captured M13 phagewith peaks representing gene VIII and gene III proteins. FIG. 22B is acontrol showing peaks representing the antibody adsorbent (singly anddoubly charged).

FIGS. 26A-26D show adsorption of M13 phage bearing an anti-tat singlechain antibody by tat protein adsorbent. Single strength is shown underphage dilutions from 1:10 to 1:10,000.

FIGS. 27A-27B show retention maps of TGF-β binding to docked TGF-βreceptor fusion protein at 1 μg/ml (FIG. 27A) and at 100 ng/ml (FIG.27B). The solid line shows binding without the presence of free TGF-βreceptor. The dashed line shows binding in the presence TGF-β receptor.

FIGS. 28 to 31 show the resolving power of retentate chromatography.FIGS. 28A-28C show resolution of proteins from Hemophilus lysate usinghydrophobic, cationic and Cu(II) adsorbents at molecular masses from 0kD to 30 kD. Each retained analyte is represented by a bar, the heightof the bar represents the intensity of the retained analyte. FIGS.29A-29C show resolution of proteins from Hemophilus lysate usinghydrophobic, cationic and Cu(II) adsorbents at molecular masses fromabout 30 kD to about 100 kD. FIG. 30 shows combined resolution from 0 kDto 30 kD of Hemophilus proteins from each of the three adsorbents. FIG.31 shows combined resolution from 20 kD to 100 kD of Hemophilus proteinsfrom each of the three adsorbents.

FIG. 32 shows the binding of GST fusion protein to a normal adsorbent.

FIGS. 33A-33B show binding of a specific ligand to GST fusion receptordocked to an adsorbent array (FIG. 33A) and lack of binding of theligand to a control array that does not include the GST fusion receptor(FIG. 33B).

DETAILED DESCRIPTION OF THE INVENTION

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

“Analyte” refers to a component of a sample which is desirably retainedand detected. The term can refer to a single component or a set ofcomponents in the sample.

“Adsorbent” refers to any material capable of adsorbing an analyte. Theterm “adsorbent” is used herein to refer both to a single material(“monoplex adsorbent”) (e.g., a compound or functional group) to whichthe analyte is exposed, and to a plurality of different materials(“multiplex adsorbent”) to which a sample is exposed. The adsorbentmaterials in a multiplex adsorbent are referred to as “adsorbentspecies.” For example, an addressable location on a substrate cancomprise a multiplex adsorbent characterized by many different adsorbentspecies (e.g., anion exchange materials, metal chelators, orantibodies), having different binding characteristics.

“Adsorb” refers to the detectable binding between an absorbent and ananalyte either before or after washing with an eluant (selectivitythreshold modifier).

“Substrate” refers to a solid phase to which an adsorbent is attached ordeposited.

“Binding characteristic” refers to a chemical and physical feature thatdictates the attraction of an adsorbent for an analyte. Two adsorbentshave different binding characteristics if, under the same elutionconditions, the adsorbents bind the same analyte with different degreesof affinity. Binding characteristics include, for example, degree ofsalt-promoted interaction, degree of hydrophobic interaction, degree ofhydrophilic interaction, degree of electrostatic interaction, and othersdescribed herein.

“Binding conditions” refer to the binding characteristics to which ananalyte is exposed.

“Eluant” refers to an agent, typically a solution, that is used tomediate adsorption of an analyte to an adsorbent. Eluants also arereferred to as “selectivity threshold modifiers.”

“Elution characteristic” refers to a feature that dictates the abilityof a particular eluant (selectivity threshold modifier) to mediateadsorption between an analyte and an absorbent. Two eluants havedifferent elution characteristics if, when put in contact with ananalyte and adsorbent, the degree of affinity of the analyte for theadsorbent differs. Elution characteristics include, for example, pH,ionic strength, modification of water structure, detergent strength,modification of hydrophobic interactions, and others described herein.

“Elution conditions” refer to the elution characteristics to which ananalyte is exposed.

“Selectivity characteristic” refers to a feature of the combination ofan adsorbent having particular binding characteristics and an eluanthaving particular elution characteristics that dictate the specificitywith which the analyte is retained to the adsorbent after washing withthe eluant.

“Selectivity conditions” refer to the selectivity characteristics towhich an analyte is exposed.

“Basis for attraction” refers to the chemical and/or physico-chemicalproperties which cause one molecule to be attracted to another.

“Strength of attraction” refers to the intensity of the attraction ofone molecule for another (also known as affinity).

“Resolve,” “resolution,” or “resolution of analyte” refers to thedetection of at least one analyte in a sample. Resolution includes thedetection of a plurality of analytes in a sample by separation andsubsequent differential detection. Resolution does not require thecomplete separation of an analyte from all other analytes in a mixture.Rather, any separation that allows the distinction between at least twoanalytes suffices.

“High information resolution” refers to resolution of an analyte in amanner that permits not only detection of the analyte, but also at leastone physico-chemical property of the analyte to be evaluated, e.g.,molecular mass.

“Desorption spectrometry” refers to a method of detecting an analyte inwhich the analyte is exposed to energy which desorbs the analyte from astationary phase into a gas phase, and the desorbed analyte or adistinguishable portion of it is directly detected by a detector,without an intermediate step of capturing the analyte on a secondstationary phase.

“Detect” refers to identifying the presence, absence or amount of theobject to be detected.

“Retention” refers to an adsorption of an analyte by an adsorbent afterwashing with an eluant.

“Retention data” refers to data indicating the detection (optionallyincluding detecting mass) of an analyte retained under a particularselectivity condition.

“Retention map” refers to a value set specifying retention data for ananalyte retained under a plurality of selectivity conditions.

“Recognition profile” refers to a value set specifying relativeretention of an analyte under a plurality of selectivity conditions.

“Complex” refers to analytes formed by the union of 2 or more analytes.

“Fragment” refers to the products of the chemical, enzymatic, orphysical breakdown of an analyte. Fragments may be in a neutral or ionicstate.

“Differential expression” refers to a detectable difference in thequalitative or quantitative presence of an analyte.

“Biological sample” refers to a sample derived from a virus, cell,tissue, organ or organism including, without limitation, cell, tissue ororgan lysates or homogenates, or body fluid samples, such as blood,urine or cerebrospinal fluid.

“Organic biomolecule” refers to an organic molecule of biologicalorigin, e.g., steroids, amino acids, nucleotides, sugars, polypeptides,polynucleotides, complex carbohydrates or lipids.

“Small organic molecule” refers to organic molecules of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes organic biopolymers (e.g., proteins, nucleic acids,etc.). Preferred small organic molecules range in size up to about 5000Da, up to about 2000 Da, or up to about 1000 Da.

“Biopolymer” refers to a polymer of biological origin, e.g.,polypeptides, polynucleotides, polysaccharides or polyglycerides (e.g.,di- or tri-glycerides).

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.The term “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides.

“Polynucleotide” refers to a polymer composed of nucleotide units.Polynucleotides include naturally occurring nucleic acids, such asdeoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well asnucleic acid analogs. Nucleic acid analogs include those which includenon-naturally occurring bases, nucleotides that engage in linkages withother nucleotides other than the naturally occurring phosphodiester bondor which include bases attached through linkages other thanphosphodiester bonds. Thus, nucleotide analogs include, for example andwithout limitation, phosphorothioates, phosphorodithioates,phosphorotriesters, phosphoramidates, boranophosphates,methylphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, ³⁵S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavadin, dioxigenin, haptens and proteins for which antiseraor monoclonal antibodies are available, or nucleic acid molecules with asequence complementary to a target. The detectable moiety oftengenerates a measurable signal, such as a radioactive, chromogenic, orfluorescent signal, that can be used to quantitate the amount of bounddetectable moiety in a sample. The detectable moiety can be incorporatedin or attached to a primer or probe either covalently, or through ionic,van der Waals or hydrogen bonds, e.g., incorporation of radioactivenucleotides, or biotinylated nucleotides that are recognized bystreptavadin. The detectable moiety may be directly or indirectlydetectable. Indirect detection can involve the binding of a seconddirectly or indirectly detectable moiety to the detectable moiety. Forexample, the detectable moiety can be the ligand of a binding partner,such as biotin, which is a binding partner for streptavadin, or anucleotide sequence, which is the binding partner for a complementarysequence, to which it can specifically hybridize. The binding partnermay itself be directly detectable, for example, an antibody may beitself labeled with a fluorescent molecule. The binding partner also maybe indirectly detectable, for example, a nucleic acid having acomplementary nucleotide sequence can be a part of a branched DNAmolecule that is in turn detectable through hybridization with otherlabeled nucleic acid molecules. (See, e.g., P D, Fahrlander and A.Klausner, Bio/Technology (1988) 6:1165.) Quantitation of the signal isachieved by, e.g., scintillation counting, densitometry, or flowcytometry.

“Plurality” means at least two.

“Purify” or “purification” means removing at least one contaminant fromthe composition to be purified. Purification does not require that thepurified compound be 100% pure.

A “ligand” is a compound that specifically binds to a target molecule.

A “receptor” is compound that specifically binds to a ligand.

“Antibody” refers to a polypeptide ligand substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically binds and recognizes an epitope (e.g., an antigen). Therecognized immunoglobulin genes include the kappa and lambda light chainconstant region genes, the alpha, gamma, delta, epsilon and mu heavychain constant region genes, and the myriad immunoglobulin variableregion genes. Antibodies exist, e.g., as intact immunoglobulins or as anumber of well characterized fragments produced by digestion withvarious peptidases. This includes, e.g., Fab′ and F(ab)′₂ fragments. Theterm “antibody,” as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies. It also includes polyclonalantibodies, monoclonal antibodies, chimeric antibodies and humanizedantibodies. “Fc” portion of an antibody refers to that portion of animmunoglobulin heavy chain that comprises one or more heavy chainconstant region domains, CH₁, CH₂ and CH₃, but does not include theheavy chain variable region.

A ligand or a receptor (e.g., an antibody) “specifically binds to” or“is specifically immunoreactive with” a compound analyte when the ligandor receptor functions in a binding reaction which is determinative ofthe presence of the analyte in a sample of heterogeneous compounds.Thus, under designated assay (e.g., immunoassay) conditions, the ligandor receptor binds preferentially to a particular analyte and does notbind in a significant amount to other compounds present in the sample.For example, a polynucleotide specifically binds under hybridizationconditions to an analyte polynucleotide comprising a complementarysequence; an antibody specifically binds under immunoassay conditions toan antigen analyte bearing an epitope against which the antibody wasraised, and an adsorbent specifically binds to an analyte under properelution conditions.

“Agent” refers to a chemical compound, a mixture of chemical compounds,a sample of undetermined composition, a combinatorial small moleculearray, a biological macromolecule, a bacteriophage peptide displaylibrary, a bacteriophage antibody (e.g., scFv) display library, apolysome peptide display library, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal cells or tissues.Suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al. (1989) Science246: 1275-1281; and Ward et al. (1989) Nature 341: 544-546. The protocoldescribed by Huse is rendered more efficient in combination with phagedisplay technology. See, e.g., Dower et al., WO 91/17271 and McCaffertyet al., WO 92/01047.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell. A host cell thatcomprises the recombinant polynucleotide is referred to as a“recombinant host cell.” The gene is then expressed in the recombinanthost cell to produce, e.g., a “recombinant polypeptide.” A recombinantpolynucleotide may serve a non-coding function (e.g., promoter, originof replication, ribosome-binding site, etc.) as well. Appropriateunicellular hosts include any of those routinely used in expressingeukaryotic or mammalian polynucleotides, including, for example,prokaryotes, such as E. coli; and eukaryotes, including for example,fungi, such as yeast; and mammalian cells, including insect cells (e.g.,Sf9) and animal cells such as CHO, R1.1, B-W, L-M, African Green MonkeyKidney cells (e.g. COS 1, COS 7, BSC 1, BSC 40 and BMT 10) and culturedhuman cells.

“Expression control sequence” refers to a nucleotide sequence in apolynucleotide that regulates the expression (transcription and/ortranslation) of a nucleotide sequence operatively linked to it.“Operatively linked” refers to a functional relationship between twoparts in which the activity of one part (e.g., the ability to regulatetranscription) results in an action on the other part (e.g.,transcription of the sequence). Expression control sequences canincludes, for example and without limitation, sequences of promoters(e.g., inducible, repressible or constitutive), enhancers, transcriptionterminators, a start codon (i.e., ATG), splicing signals for introns,and stop codons.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and virusesthat incorporate the recombinant polynucleotide.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Energy absorbing molecule” refers to refers to a molecule that absorbsenergy from an energy source in a desorption spectrometer therebyenabling desorption of analyte from a probe surface. Energy absorbingmolecules used in MALDI are frequently referred to as “matrix.” Cinnamicacid derivatives, cinapinic acid and dihydroxybenzoic acid arefrequently used as energy absorbing molecules in laser desorption ofbioorganic molecules.

II. RETENTATE CHROMATOGRAPHY

Retentate chromatography is a method for the multidimensional resolutionof analytes in a sample. The method involves (1) selectively adsorbinganalytes from a sample to a substrate under a plurality of differentadsorbent/eluant combinations (“selectivity conditions”) and (2)detecting the retention of adsorbed analytes by desorption spectrometry.Each selectivity condition provides a first dimension of separation,separating adsorbed analytes from those that are not adsorbed.Desorption mass spectrometry provides a second dimension of separation,separating adsorbed analytes from each other according to mass. Becauseretentate chromatography involves using a plurality of differentselectivity conditions, many dimensions of separation are achieved. Therelative adsorption of one or more analytes under the two selectivityconditions also can be determined. This multidimensional separationprovides both resolution of the analytes and their characterization.

Further, the analytes thus separated remain docked in a retentate mapthat is amenable to further manipulation to examine, for example,analyte structure and/or function. Also, the docked analytes can,themselves, be used as adsorbents to dock other analytes exposed to thesubstrate. In sum, the present invention provides a rapid,multidimensional and high information resolution of analytes.

The method can take several forms. In one embodiment, the analyte isadsorbed to two different adsorbents at two physically differentlocations and each adsorbent is washed with the same eluant (selectivitythreshold modifier). In another embodiment, the analyte is adsorbed tothe same adsorbent at two physically different locations and washed withtwo different eluants. In another embodiment, the analyte is adsorbed totwo different adsorbents in physically different locations and washedwith two different eluants. In another embodiment, the analyte isadsorbed to an adsorbent and washed with a first eluant, and retentionis detected; then, the adsorbed analyte is washed with a second,different eluant, and subsequent retention is detected.

A. Methods of Performing Retentate Chromatography

1. Exposing the Analyte to Selectivity Conditions

a. Substrate Preparation

In performing retentate chromatography an analyte that is retained by anadsorbent is presented to an energy source on a substrate. A samplecontaining the analyte may be contacted to the adsorbent before or afterthe adsorbent is affixed to the substrate that will serve to present theanalyte to the desorption means. For contacting purposes, the adsorbentmay be in liquid form or solid form (i.e., on a substrate or solidphase). Specifically, the adsorbent may be in the form of a solution,suspension, dispersion, water-in-oil emulsion, oil-in-water emulsion, ormicroemulsion. When the adsorbent is provided in the form of asuspension, dispersion, emulsion or microemulsion, a suitable surfactantmay also be present. In this embodiment, the sample may be contactedwith the adsorbent by admixing a liquid sample with the liquidadsorbent. Alternatively, the sample may be provided on a solid supportand contacting will be accomplished by bathing, soaking, or dipping thesample-containing solid support in the liquid adsorbent. In addition,the sample may be contacted by spraying or washing over the solidsupport with the liquid adsorbent. In this embodiment, differentadsorbents may be provided in different containers.

In one embodiment, the adsorbent is provided on a substrate. Thesubstrate can be any material which is capable of binding or holding theadsorbent. Typically, the substrate is comprised of glass; ceramic;electrically conducting polymers (e.g. carbonized PEEK(polyetheretherketone)); TEFLON® (polytetrafluoroethylene) coatedmaterials; organic polymers; native biopolymers; metals (e.g., nickel,brass, steel or aluminum); films; porous and non-porous beads ofcross-linked polymers (e.g., agarose, cellulose or dextran); otherinsoluble polymers; or combinations thereof.

In one embodiment, the substrate takes the form of a probe or a samplepresenting means that is inserted into a desorption detector. Forexample, referring to FIG. 1, the substrate can take the form of astrip. The adsorbent can be attached to the substrate in the form of alinear array of spots, each of which can be exposed to the analyte.Several strips can be joined together so that the plurality ofadsorbents form an array 30 having discrete spots in defined rows. Thesubstrate also can be in the form of a plate having an array ofhorizontal and vertical rows of adsorbents which form a regulargeometric pattern such as a square, rectangle or circle.

Probes can be produced as follows. The substrate can be any solidmaterial, for example, stainless steel, aluminum or a silicon wafer. Ametal substrate can then be coated with a material that allowsderivitization of the surface. For example a metal surface can be coatedwith silicon oxide, titanium oxide or gold.

The surface is then derivatized with a bifunctional linker. The linkerincludes at one end a functional group that can covalently bind with afunctional group on the surface. Thus the functional group can be aninorganic oxide or a sulfhydryl group for gold. The other end of thelinker generally has an amino functionality. Useful bifunctional linkersinclude aminopropyl triethoxysilane or aminoethyl disulfide.

Once bound to the surface, the linkers are further derivatized withgroups that function as the adsorbent. Generally the adsorbent is addedto addressable locations on the probe. In one type of probe spots ofabout 3 mm in diameter are arrange in an orthogonal array. Theadsorbents can, themselves, be part of bifunctional molecules containinga group reactive with the available amino group and the functional groupthat acts as the adsorbent. Functional groups include, for example,normal phase (silicon oxide), reverse phase (C₁₈ aliphatic hydrocarbon),quaternary amine and sulphonate. Also, the surface can be furtherderivatized with other bifunctional molecules such as carbodiimide andN-hydroxysuccinimide, creating a pre-activated blank. These blanks canbe functionalized with bioorganic adsorbents (e.g., nucleic acids,antibodies and other protein ligands). Biopolymers can bind thefunctional groups on the blanks through amine residues or sulfhydrylresidues. In one embodiment, the adsorbents are bound to cross-linkedpolymers (e.g., films) that are themselves bound to the surface of theprobe through the available functional groups. Such polymers include,for example, cellulose, dextran, carboxymethyl dextran, polyacrylamideand mixtures of these. Probes with attached adsorbents are ready foruse.

In another embodiment, the adsorbent is attached to a first substrate toprovide a solid phase, such as a polymeric or glass bead, which issubsequently positioned on a second substrate which functions as themeans for presenting the sample to the desorbing energy of thedesorption detector. For example, the second substrate can be in theform of a plate having a series of wells at predetermined addressablelocations. The wells can function as containers for a first substratederivatized with the adsorbent, e.g., polymeric beads derivatized withthe adsorbent. One advantage of this embodiment is that the analyte canbe adsorbed to the first substrate in one physical context, andtransferred to the sample presenting substrate for analysis bydesorption spectrometry.

Typically, the substrate is adapted for use with the detectors employedin the methods of the present invention for detecting the analyte boundto and retained by the adsorbent. In one embodiment, the substrate isremovably insertable into a desorption detector where an energy sourcecan strike the spot and desorb the analyte. The substrate can besuitable for mounting in a horizontally and/or vertically translatablecarriage that horizontally and/or vertically moves the substrate tosuccessively position each predetermined addressable location ofadsorbent in a path for interrogation by the energy source and detectionof the analyte bound thereto. The substrate can be in the form of aconventional mass spectrometry probe.

The strips, plates, or probes of substrate can be produced usingconventional techniques. Thereafter, the adsorbent can be directly orindirectly coupled, fitted, or deposited on the substrate prior tocontacting with the sample containing the analyte. The adsorbent may bedirectly or indirectly coupled to the substrate by any suitable means ofattachment or immobilization. For example, the adsorbent can be directlycoupled to the substrate by derivatizing the substrate with theadsorbent to directly bind the adsorbent to the substrate throughcovalent or non-covalent bonding.

Attachment of the adsorbent to the substrate can be accomplished througha variety of mechanisms. The substrate can be derivatized with a fullyprepared adsorbent molecule by attaching the previously preparedadsorbent molecule to the substrate. Alternatively, the adsorbent can beformed on the substrate by attaching a precursor molecule to thesubstrate and subsequently adding additional precursor molecules to thegrowing chain bound to the substrate by the first precursor molecule.This mechanism of building the adsorbent on the substrate isparticularly useful when the adsorbent is a polymer, particularly abiopolymer such as a DNA or RNA molecule. A biopolymer adsorbent can beprovided by successively adding bases to a first base attached to thesubstrate using methods known in the art of oligonucleotide chiptechnology. See, e.g., U.S. Pat. No. 5,445,934 (Foder et al.).

As can be seen from FIG. 2, as few as two and as many as 10, 100, 1000,10,000 or more adsorbents can be coupled to a single substrate. The sizeof the adsorbent site may be varied, depending on experimental designand purpose. However, it need not be larger than the diameter of theimpinging energy source (e.g., laser spot diameter). The spots cancontinue the same or different adsorbents. In some cases, it isadvantageous to provide the same adsorbent at multiple locations on thesubstrate to permit evaluation against a plurality of different eluantsor so that the bound analyte can be preserved for future use orreference, perhaps in secondary processing. By providing a substratewith a plurality of different adsorbents, it is possible to utilize theplurality of binding characteristics provided by the combination ofdifferent adsorbents with respect to a single sample and thereby bindand detect a wider variety of different analytes. The use of a pluralityof different adsorbents on a substrate for evaluation of a single sampleis essentially equivalent to concurrently conducting multiplechromatographic experiments, each with a different chromatographycolumn, but the present method has the advantage of requiring only asingle system.

When the substrate includes a plurality of adsorbents, it isparticularly useful to provide the adsorbents in predeterminedaddressable locations. By providing the adsorbents in predeterminedaddressable locations, it is possible to wash an adsorbent at a firstpredetermined addressable location with a first eluant and to wash anadsorbent at a second predetermined addressable location with a secondeluant. In this manner, the binding characteristics of a singleadsorbent for the analyte can be evaluated in the presence of multipleeluants which each selectively modify the binding characteristics of theadsorbent in a different way. The addressable locations can be arrangedin any pattern, but preferably in regular patterns, such as lines,orthogonal arrays, or regular curves, such as circles. Similarly, whenthe substrate includes a plurality of different adsorbents, it ispossible to evaluate a single eluant with respect to each differentadsorbent in order to evaluate the binding characteristics of a givenadsorbent in the presence of the eluant. It is also possible to evaluatethe binding characteristics of different adsorbents in the presence ofdifferent eluants.

(1) Incremental or Gradient Adsorbent Surfaces

A series of adsorbents having different binding characteristics can beprovided by synthesizing a plurality of different polymeric adsorbentson the substrate. The different polymeric adsorbents can be provided byattaching a precursor molecule to the substrate, initializing thepolymerization reaction, and terminating the polymerization reaction atvaried degrees of completion for each adsorbent. Also, the terminalfunctional groups in the polymers can be reacted so as to chemicallyderivatize them to varying degrees with different affinity reagent(e.g., —NH₃, or COO⁻). By terminating the polymerization orderivatization reaction, adsorbents of varying degrees of polymerizationor derivatization are produced. The varying degrees of polymerization orderivatization provide different binding characteristics for eachdifferent polymeric adsorbent. This embodiment is particularly usefulfor providing a plurality of different biopolymer adsorbents on asubstrate.

If desired, the polymerization reactions can be carried out in areaction vessel, rather than on the substrate itself. For example,polymeric adsorbents of varying binding characteristics can be providedby extracting an aliquot of product from the reaction vessel as thepolymerization/derivatization reaction is proceeding. The aliquots,having been extracted at various points during thepolymerization/derivatization reaction will exhibit varied degrees ofpolymerization/derivatization to yield a plurality of differentadsorbents. The different aliquots of product can then be utilized asadsorbents having different binding characteristics. Alternatively, aplurality of different adsorbents can be provided by sequentiallyrepeating the steps of terminating the reaction, withdrawing an aliquotof product, and re-starting the polymerization/derivatization reaction.The products extracted at each termination point will exhibit varyingdegrees of polymerization/derivatization and as a result will provide aplurality of adsorbents having different binding characteristics.

In one embodiment, a substrate is provided in the form of a strip or aplate that is coated with adsorbent in which one or more bindingcharacteristic varies in a one- or two-dimensional gradient. Forexample, a strip is provided having an adsorbent that is weaklyhydrophobic at one end and strongly hydrophobic at the other end. Or, aplate is provided that is weakly hydrophobic and anionic in one corner,and strongly hydrophobic and anionic in the diagonally opposite corner.Such adsorption gradients are useful in the qualitative analysis of ananalyte. Adsorption gradients can be made by a controlled sprayapplication or by flowing material across a surface in a time-wisemanner to allow incremental completion of a reaction over the dimensionof the gradient. This process can be repeated, at right angles, toprovide orthogonal gradients of similar or different adsorbents withdifferent binding characteristics.

The sample containing the analyte may be contacted to the adsorbenteither before or after the adsorbent is positioned on the substrateusing any suitable method which will enable binding between the analyteand the adsorbent. The adsorbent can simply be admixed or combined withthe sample. The sample can be contacted to the adsorbent by bathing orsoaking the substrate in the sample, or dipping the substrate in thesample, or spraying the sample onto the substrate, by washing the sampleover the substrate, or by generating the sample or analyte in contactwith the adsorbent. In addition, the sample can be contacted to theadsorbent by solubilizing the sample in or admixing the sample with aneluant and contacting the solution of eluant and sample to the adsorbentusing any of the foregoing techniques (i.e., bathing, soaking, dipping,spraying, or washing over).

b. Contacting the Analyte to the Adsorbent

Exposing the sample to an eluant prior to binding the analyte to theadsorbent has the effect of modifying the selectivity of the adsorbentwhile simultaneously contacting the sample to the adsorbent. Thosecomponents of the sample which will bind to the adsorbent and thereby beretained will include only those components which will bind theadsorbent in the presence of the particular eluant which has beencombined with the sample, rather than all components which will bind tothe adsorbent in the absence of elution characteristics which modify theselectivity of the adsorbent.

The sample should be contacted to the adsorbent for a period of timesufficient to allow the analyte to bind to the adsorbent. Typically, thesample is contacted with the analyte for a period of between about 30seconds and about 12 hours. Preferably, the sample is contacted to theanalyte for a period of between about 30 seconds and about 15 minutes.

The temperature at which the sample is contacted to the adsorbent is afunction of the particular sample and adsorbents selected. Typically,the sample is contacted to the adsorbent under ambient temperature andpressure conditions, however, for some samples, modified temperature(typically 4° C. through 37° C.) and pressure conditions can bedesirable and will be readily determinable by those skilled in the art.

Another advantage of the present invention over conventional detectiontechniques is that the present invention enables the numerous differentexperiments to be conducted on a very small amount of sample. Generally,a volume of sample containing from a few atommoles to 100 picomoles ofanalyte in about 1 μl to 500 μl is sufficient for binding to theadsorbent. Analyte may be preserved for future experiments after bindingto the adsorbent because any adsorbent locations which are not subjectedto the steps of desorbing and detecting all of the retained analyte willretain the analyte thereon. Therefore, in the case where only a verysmall fraction of sample is available for analysis, the presentinvention provides the advantage of enabling a multitude of experimentswith different adsorbents and/or eluants to be carried out at differenttimes without wasting sample.

c. Washing the Adsorbent with Eluants

After the sample is contacted with the analyte, resulting in the bindingof the analyte to the adsorbent, the adsorbent is washed with eluant.Typically, to provide a multi-dimensional analysis, each adsorbentlocation is washed with at least a first and a second different eluants.Washing with the eluants modifies the analyte population retained on aspecified adsorbent. The combination of the binding characteristics ofthe adsorbent and the elution characteristics of the eluant provide theselectivity conditions which control the analytes retained by theadsorbent after washing. Thus, the washing step selectively removessample components from the adsorbent.

The washing step can be carried out using a variety of techniques. Forexample, as seen above, the sample can be solubilized in or admixed withthe first eluant prior to contacting the sample to the adsorbent.Exposing the sample to the first eluant prior to or simultaneously withcontacting the sample to the adsorbent has, to a first approximation,the same net effect as binding the analyte to the adsorbent andsubsequently washing the adsorbent with the first eluant. After thecombined solution is contacted to the adsorbent, the adsorbent can bewashed with the second or subsequent eluants.

Washing an adsorbent having the analyte bound thereto can beaccomplished by bathing, soaking, or dipping the substrate having theadsorbent and analyte bound thereon in an eluant; or by rinsing,spraying, or washing over the substrate with the eluant. Theintroduction of eluant to small diameter spots of affinity reagent isbest achieved by a microfluidics process.

When the analyte is bound to adsorbent at only one location and aplurality of different eluants are employed in the washing step,information regarding the selectivity of the adsorbent in the presenceof each eluant individually may be obtained. The analyte bound toadsorbent at one location may be determined after each washing witheluant by following a repeated pattern of washing with a first eluant,desorbing and detecting retained analyte, followed by washing with asecond eluant, and desorbing and detecting retained analyte. The stepsof washing followed by desorbing and detecting can be sequentiallyrepeated for a plurality of different eluants using the same adsorbent.In this manner the adsorbent with retained analyte at a single locationmay be reexamined with a plurality of different eluants to provide acollection of information regarding the analytes retained after eachindividual washing.

The foregoing method is also useful when adsorbents are provided at aplurality of predetermined addressable locations, whether the adsorbentsare all the same or different. However, when the analyte is bound toeither the same or different adsorbents at a plurality of locations, thewashing step may alternatively be carried out using a more systematicand efficient approach involving parallel processing. Namely, the stepof washing can be carried out by washing an adsorbent at a firstlocation with eluant, then washing a second adsorbent with eluant, thendesorbing and detecting the analyte retained by the first adsorbent andthereafter desorbing and detecting analyte retained by the secondadsorbent. In other words, all of the adsorbents are washed with eluantand thereafter analyte retained by each is desorbed and detected foreach location of adsorbent. If desired, after detection at eachadsorbent location, a second stage of washings for each adsorbentlocation may be conducted followed by a second stage of desorption anddetection. The steps of washing all adsorbent locations, followed bydesorption and detection at each adsorbent location can be repeated fora plurality of different eluants. In this manner, an entire array may beutilized to efficiently determine the character of analytes in a sample.The method is useful whether all adsorbent locations are washed with thesame eluant in the first washing stage or whether the plurality ofadsorbents are washed with a plurality of different eluants in the firstwashing stage.

2. Detection

Analytes retained by the adsorbent after washing are adsorbed to thesubstrate. Analytes retained on the substrate are detected by desorptionspectrometry: desorbing the analyte from the adsorbent and directlydetecting the desorbed analytes.

a. Methods for Desorption

Desorbing the analyte from the adsorbent involves exposing the analyteto an appropriate energy source. Usually this means striking the analytewith radiant energy or energetic particles. For example, the energy canbe light energy in the form of laser energy (e.g., UV laser) or energyfrom a flash lamp. Alternatively, the energy can be a stream of fastatoms. Heat may also be used to induce/aid desorption.

Methods of desorbing and/or ionizing analytes for direct analysis arewell known in the art. One such method is called matrix-assisted laserdesorption/ionization, or MALDI. In MALDI, the analyte solution is mixedwith a matrix solution and the mixture is allowed to crystallize afterbeing deposited on an inert probe surface, trapping the analyte withinthe crystals may enable desorption. The matrix is selected to absorb thelaser energy and apparently impart it to the analyte, resulting indesorption and ionization. Generally, the matrix absorbs in the UVrange. MALDI for large proteins is described in, e.g., U.S. Pat. No.5,118,937 (Hillenkamp et al.) and U.S. Pat. No. 5,045,694 (Beavis andChait).

Surface-enhanced laser desorption/ionization, or SELDI, represents asignificant advance over MALDI in terms of specificity, selectivity andsensitivity. SELDI is described in U.S. Pat. No. 5,719,060 (Hutchens andYip). SELDI is a solid phase method for desorption in which the analyteis presented to the energy stream on a surface that enhances analytecapture and/or desorption. In contrast, MALDI is a liquid phase methodin which the analyte is mixed with a liquid material that crystallizesaround the analyte.

One version of SELDI, called SEAC (Surface-Enhanced Affinity Capture),involves presenting the analyte to the desorbing energy in associationwith an affinity capture device (i.e., an adsorbent). It was found thatwhen an analyte is so adsorbed, it can be presented to the desorbingenergy source with a greater opportunity to achieve desorption of thetarget analyte. An energy absorbing material can be added to the probeto aid desorption. Then the probe is presented to the energy source fordesorbing the analyte.

Another version of SELDI, called SEND (Surface-Enhanced NeatDesorption), involves the use of a layer of energy absorbing materialonto which the analyte is placed. A substrate surface comprises a layerof energy absorbing molecules chemically bond to the surface and/oressentially free of crystals. Analyte is then applied alone (i.e., neat)to the surface of the layer, without being substantially mixed with it.The energy absorbing molecules, as do matrix, absorb the desorbingenergy and cause the analyte to be desorbed. This improvement issubstantial because analytes can now be presented to the energy sourcein a simpler and more homogeneous manner because the performance ofsolution mixtures and random crystallization is eliminated. Thisprovides more uniform and predictable results that enable automation ofthe process. The energy absorbing material can be classical matrixmaterial or can be matrix material whose pH has been neutralized orbrought into the basic range. The energy absorbing molecules can bebound to the probe through covalent or noncovalent means.

Another version of SELDI, called SEPAR (Surface-Enhanced PhotolabileAttachment and Release), involves the use of photolabile attachmentmolecules. A photolabile attachment molecule is a divalent moleculehaving one site covalently bound to a solid phase, such a flat probesurface or another solid phase, such as a bead, that can be made part ofthe probe, and a second site that can be covalently bound with theaffinity reagent or analyte. The photolabile attachment molecule, whenbound to both the surface and the analyte, also contains a photolabilebond that can release the affinity reagent or analyte upon exposure tolight. The photolabile bond can be within the attachment molecule or atthe site of attachment to either the analyte (or affinity reagent) orthe probe surface.

b. Method For Direct Detection of Analytes

The desorbed analyte can be detected by any of several means. When theanalyte is ionized in the process of desorption, such as in laserdesorption/ionization mass spectrometry, the detector can be an iondetector. Mass spectrometers generally include means for determining thetime-of-flight of desorbed ions. This information is converted to mass.However, one need not determine the mass of desorbed ions to resolve anddetect them: the fact that ionized analytes strike the detector atdifferent times provides detection and resolution of them.

Alternatively, the analyte can be detectably labeled with, e.g., afluorescent moiety or with a radioactive moiety. In these cases, thedetector can be a fluorescence or radioactivity detector.

A plurality of detection means can be implemented in series to fullyinterrogate the analyte components and function associated withretentate at each location in the array.

c. Desorption Detectors

Desorption detectors comprise means for desorbing the analyte from theadsorbent and means for directly detecting the desorbed analyte. Thatis, the desorption detector detects desorbed analyte without anintermediate step of capturing the analyte in another solid phase andsubjecting it to subsequent analysis. Detection of an analyte normallywill involve detection of signal strength. This, in turn, reflects thequantity of analyte adsorbed to the adsorbent.

Beyond these two elements, the deposition detector also can have otherelements. One such element is means to accelerate the desorbed analytetoward the detector. Another element is means for determining thetime-of-flight of analyte from desorption to detection by the detector.

A preferred desorption detector is a laser desorption/ionization massspectrometer, which is well known in the art. The mass spectrometerincludes a port into which the substrate that carries the adsorbedanalyte, e.g., a probe, is inserted. Desorption is accomplished bystriking the analyte with energy, such as laser energy. The device caninclude means for translating the surface so that any spot on the arrayis brought into line with the laser beam. Striking the analyte with thelaser results in desorption of the intact analyte into the flight tubeand its ionization. The flight tube generally defines a vacuum space.Electrified plates in a portion of the vacuum tube create an electricalpotential which accelerate the ionized analyte toward the detector. Aclock measures the time of flight and the system electronics determinesvelocity of the analyte and converts this to mass. As any person skilledin the art understands, any of these elements can be combined with otherelements described herein in the assembly of desorption detectors thatemploy various means of desorption, acceleration, detection, measurementof time, etc.

B. Selectivity Conditions

One advantage of the invention is the ability to expose the analytes toa variety of different binding and elution conditions, thereby providingboth increased resolution of analytes and information about them in theform of a recognition profile. As in conventional chromatographicmethods, the ability of the adsorbent to retain the analyte is directlyrelated to the attraction or affinity of the analyte for the adsorbentas compared to the attraction or affinity of the analyte for the eluantor the eluant for the adsorbent. Some components of the sample may haveno affinity for the adsorbent and therefore will not bind to theadsorbent when the sample is contacted to the adsorbent. Due to theirinability to bind to the adsorbent, these components will be immediatelyseparated from the analyte to be resolved. However, depending upon thenature of the sample and the particular adsorbent utilized, a number ofdifferent components can initially bind to the adsorbent.

1. Adsorbents

Adsorbents are the materials that bind analytes. A plurality ofadsorbents can be employed in retentate chromatography. Differentadsorbents can exhibit grossly different binding characteristics,somewhat different binding characteristics, or subtly different bindingcharacteristics. Adsorbents which exhibit grossly different bindingcharacteristics typically differ in their bases of attraction or mode ofinteraction. The basis of attraction is generally a function of chemicalor biological molecular recognition. Bases for attraction between anadsorbent and an analyte include, for example, (1) a salt-promotedinteraction, e.g., hydrophobic interactions, thiophilic interactions,and immobilized dye interactions; (2) hydrogen bonding and/or van derWaals forces interactions and charge transfer interactions, such as inthe case of a hydrophilic interactions; (3) electrostatic interactions,such as an ionic charge interaction, particularly positive or negativeionic charge interactions; (4) the ability of the analyte to formcoordinate covalent bonds (i.e., coordination complex formation) with ametal ion on the adsorbent; (5) enzyme-active site binding; (6)reversible covalent interactions, for example, disulfide exchangeinteractions; (7) glycoprotein interactions; (8) biospecificinteractions; or (9) combinations of two or more of the foregoing modesof interaction. That is, the adsorbent can exhibit two or more bases ofattraction, and thus be known as a “mixed functionality” adsorbent.

a. Salt-promoted Interaction Adsorbents

Adsorbents which are useful for observing salt-promoted interactionsinclude hydrophobic interaction adsorbents. Examples of hydrophobicinteraction adsorbents include matrices having aliphatic hydrocarbons,specifically C₁-C₁₈ aliphatic hydrocarbons; and matrices having aromatichydrocarbon functional groups such as phenyl groups. Hydrophobicinteraction adsorbents bind analytes which include uncharged solventexposed amino acid residues, and specifically amino acid residues whichare commonly referred to as nonpolar, aromatic and hydrophobic aminoacid residues, such as phenylalanine and tryptophan. Specific examplesof analytes which will bind to a hydrophobic interaction adsorbentinclude lysozyme and DNA. Without wishing to be bound by a particulartheory, it is believed that DNA binds to hydrophobic interactionadsorbents by the aromatic nucleotides in DNA, specifically, the purineand pyrimidine groups.

Another adsorbent useful for observing salt-promoted interactionsincludes thiophilic interaction adsorbents, such as for example T-GEL®(thiophilic gel) which is one type of thiophilic adsorbent commerciallyavailable from Pierce, Rockford, Ill. Thiophilic interaction adsorbentsbind, for example, immunoglobulins such as IgG. The mechanism ofinteraction between IgG and T-GEL® is not completely known, but solventexposed trp residues are suspected to play a role.

A third adsorbent which involves salt-promoted ionic interactions andalso hydrophobic interactions includes immobilized dye interactionadsorbents. Immobilized dye interaction adsorbents include matrices ofimmobilized dyes such as for example CIBACHRON™ C₂₉H₂₀CIN₉O₁₁S₂ blueavailable from Pharmacia Biotech, Piscataway, N.J. Immobilized dyeinteraction adsorbents bind proteins and DNA generally. One specificexample of a protein which binds to an immobilized dye interactionadsorbent is bovine serum albumin (BSA).

b. Hydrophilic Interaction Adsorbents

Adsorbents which are useful for observing hydrogen bonding and/or vander Waals forces on the basis of hydrophilic interactions includesurfaces comprising normal phase adsorbents such as silicon-oxide (i.e.,glass). The normal phase or silicon-oxide surface, acts as a functionalgroup. In addition, adsorbents comprising surfaces modified withhydrophilic polymers such as polyethylene glycol, dextran, agarose, orcellulose can also function as hydrophilic interaction adsorbents. Mostproteins will bind hydrophilic interaction adsorbents because of a groupor combination of amino acid residues (i.e., hydrophilic amino acidresidues) that bind through hydrophilic interactions involving hydrogenbonding or van der Waals forces. Examples of proteins which will bindhydrophilic interaction adsorbents include myoglobin, insulin andcytochrome C.

In general, proteins with a high proportion of polar or charged aminoacids will be retained on a hydrophilic surface. Alternatively,glycoproteins with surface exposed hydrophilic sugar moieties, also havehigh affinity for hydrophilic adsorbents.

c. Electrostatic Interaction Adsorbents

Adsorbents which are useful for observing electrostatic or ionic chargeinteractions include anionic adsorbents such as, for example, matricesof sulfate anions (i.e., SO₃ ⁻) and matrices of carboxylate anions(i.e., COO⁻) or phosphate anions (OPO₃ ⁻). Matrices having sulfateanions are permanent negatively charged. However, matrices havingcarboxylate anions have a negative charge only at a pH above their pKa.At a pH below the pKa, the matrices exhibit a substantially neutralcharge. Suitable anionic adsorbents also include anionic adsorbentswhich are matrices having a combination of sulfate and carboxylateanions and phosphate anions. The combination provides an intensity ofnegative charge that can be continuously varied as a function of pH.These adsorbents attract and bind proteins and macromolecules havingpositive charges, such as for example ribonuclease and lactoferrin.Without washing to be bound by a particular theory, it is believed thatthe electrostatic interaction between an adsorbent and positivelycharged amino acid residues including lysine residues, arginineresidues, and histidyl residues are responsible for the bindinginteraction.

Other adsorbents which are useful for observing electrostatic or ioniccharge interactions include cationic adsorbents. Specific examples ofcationic adsorbents include matrices of secondary, tertiary orquaternary amines. Quaternary amines are permanently positively charged.However, secondary and tertiary amines have charges that are pHdependent. At a pH below the pKa, secondary and tertiary amines arepositively charged, and at a pH above their pKa, they are negativelycharged. Suitable cationic adsorbents also include cationic adsorbentswhich are matrices having combinations of different secondary, tertiary,and quaternary amines. The combination provides an intensity of positivecharge that can be continuously varied as a function of pH. Cationicinteraction adsorbents bind anionic sites on molecules includingproteins having solvent exposed amino acid residues, such as asparticacid and glutamic acid residues.

In the case of ionic interaction adsorbents (both anionic and cationic)it is often desirable to use a mixed mode ionic adsorbent containingboth anions and cations. Such adsorbents provide a continuous bufferingcapacity as a function of pH. The continuous buffering capacity enablesthe exposure of a combination of analytes to eluants having differingbuffering components especially in the pH range of from 2 to 11. Thisresults in the generation of local pH environments on the adsorbentwhich are defined by immobilized titratable proton exchange groups. Suchsystems are equivalent to the solid phase separation technique known aschromatofocusing. Follicle stimulating hormone isoforms, which differmainly in the charged carbohydrate components are separated on achromatofocusing adsorbent.

Still other adsorbents which are useful for observing electrostaticinteractions include dipole-dipole interaction adsorbents in which theinteractions are electrostatic but no formal charge or titratableprotein donor or acceptor is involved.

d. Coordinate Covalent Interaction Adsorbents

Adsorbents which are useful for observing the ability to form coordinatecovalent bonds with metal ions include matrices bearing, for example,divalent and trivalent metal ions. Matrices of immobilized metal ionchelators provide immobilized synthetic organic molecules that have oneor more electron donor groups which form the basis of coordinatecovalent interactions with transition metal ions. The primary electrondonor groups functioning as immobilized metal ion chelators includeoxygen, nitrogen, and sulfur. The metal ions are bound to theimmobilized metal ion chelators resulting in a metal ion complex havingsome number of remaining sites for interaction with electron donorgroups on the analyte. Suitable metal ions include in general transitionmetal ions such as copper, nickel, cobalt, zinc, iron, and other metalions such as aluminum and calcium. Without using to be bound by anyparticular theory, metals ions are believed to interact selectively withspecific amino acid residues in peptides, proteins, or nucleic acids.Typically, the amino acid residues involved in such interactions includehistidine residues, tyrosine residues, tryptophan residues, cysteineresidues, and amino acid residues having oxygen groups such as asparticacid and glutamic acid. For example, immobilized ferric ions interactwith phosphoserine, phosphotyrosine, and phosphothreonine residues onproteins. Depending on the immobilized metal ion, only those proteinswith sufficient local densities of the foregoing amino acid residueswill be retained by the adsorbent. Some interactions between metal ionsand proteins can be so strong that the protein cannot be served from thecomplex by conventional means. Human β casein, which is highlyphosphorylated, binds very strongly to immobilized Fe(III). Recombinantproteins which are expressed with a 6 Histidine tag, binds very stronglyto immobilized Cu(II) and Ni(II).

e. Enzyme-Active Site Interaction Adsorbents

Adsorbents which are useful for observing enzyme-active site bindinginteractions include proteases (such as trypsin), phosphatases, kinases,and nucleases. The interaction is a sequence-specific interaction of theenzyme binding site on the analyte (typically a biopolymer) with thecatalytic binding site on the enzyme. Enzyme binding sites of this typeinclude, for example, active sites of trypsin interacting with proteinsand peptides having lysine-lysine or lysine-arginine pairs in theirsequence. More specifically, soybean trypsin inhibitor interacts withand binds to an adsorbent of immobilized trypsin. Alternatively, serineproteases are selectively retained on immobilized L-arginine adsorbent.

f. Reversible Covalent Interaction Adsorbents

Adsorbents which are useful for observing reversible covalentinteractions include disulfide exchange interaction adsorbents.Disulfide exchange interaction adsorbents include adsorbents comprisingimmobilized sulfhydryl groups, e.g., mercaptoethanol or immobilizeddithiothrietol. The interaction is based upon the formation of covalentdisulfide bonds between the adsorbent and solvent exposed cysteineresidues on the analyte. Such adsorbents bind proteins or peptideshaving cysteine residues and nucleic acids including bases modified tocontain reduced sulfur compounds.

g. Glycoprotein Interaction Adsorbents

Adsorbents which are useful for observing glycoprotein interactionsinclude glycoprotein interaction adsorbents such as adsorbents havingimmobilize lectins (i.e., proteins bearing oligosaccharides) therein, anexample of which is CONCONAVALIN™, which is commercially available fromPharmacia Biotech of Piscataway, N.J. Such adsorbents function on thebasis of the interaction involving molecular recognition of carbohydratemoieties on macromolecules. Examples of analytes which interact with andbind to glycoprotein interaction adsorbents include glycoproteins,particularly histidine-rich glycoproteins, whole cells and isolatedsubcellular fractions.

h. Biospecific Interaction Adsorbents

Adsorbents which are useful for observing biospecific interactions aregenerically termed “biospecific affinity adsorbents.” Adsorption isconsidered biospecific it if is selective and the affinity (equilibriumdissociation constant, Kd) is at least 10⁻³ M to (e.g., 10⁻⁵ M, 10⁻⁷ M,10⁻⁹ M). Examples of biospecific affinity adsorbents include anyadsorbent which specifically interacts with and binds a particularbiomolecule. Biospecific affinity adsorbents include for example,immobilized antibodies which bind to antigens; immobilized DNA whichbinds to DNA binding proteins, DNA, and RNA; immobilized substrates orinhibitors which bind to proteins and enzymes; immobilized drugs whichbind to drug binding proteins; immobilized ligands which bind toreceptors; immobilized receptors which bind to ligands; immobilized RNAwhich binds to DNA and RNA binding proteins; immobilized avidin orstreptavidin which bind biotin and biotinylated molecules; immobilizedphospholipid membranes and vesicles which bind lipid-binding proteins.Enzymes are useful adsorbents that can modify an analyte adsorbentthereto. Cells are useful as adsorbents. Their surfaces present complexbinding characteristics. Adsorption to cells is useful for identifying,e.g., ligands or signal molecules that bind to surface receptors.Viruses or phage also are useful as adsorbents. Viruses frequently haveligands for cell surfaces receptors (e.g., gp120 for CD4). Also, in theform a phage display library, phage coat proteins act as agents fortesting binding to targets. Biospecific interaction adsorbents rely onknown specific interactions such as those described above. Otherexamples of biospecific interactions for which adsorbents can beutilized will be readily apparent to those skilled in the art and arecontemplated by the present invention.

In one embodiment, the biospecific adsorbent can further comprise anauxiliary, or “helper”, molecule that does not directly participate inbinding the target analyte.

i. Degrees of Binding Specificity

By exposure to adsorbents having different modes of interaction, thecomponents of a sample can be grossly divided based upon theirinteraction with the different adsorbents. Thus, the attraction of theanalyte for adsorbents having different modes of interaction provides afirst separation parameter. For example, by exposing a sample containingthe analyte to a first adsorbent with a basis of attraction involvinghydrophobicity and a second adsorbent with a basis of attractioninvolving ionic charge, it is possible to separate from the sample thoseanalytes which bind to a hydrophobic adsorbent and to separate thoseanalytes which bind to an adsorbent having the particular ionic charge.

Adsorbents having different bases of attraction provide resolution ofthe analyte with a low degree of specificity because the adsorbent willbind not only the analyte, but any other component in the sample whichalso exhibits an attraction for the adsorbent by the same basis ofattraction. For example, a hydrophobic adsorbent will bind not only ahydrophobic analyte, but also any other hydrophobic components in thesample; a negatively charged adsorbent will bind not only a positivelycharged analyte, but also any other positively charged component in thesample; and so on.

The resolution of analytes based upon the basis of attraction of theanalyte for the adsorbent can be further refined by exploiting bindingcharacteristics of relatively intermediate specificity or alteredstrength of attraction. Resolution of the analyte on the basis ofbinding characteristics of intermediate specificity can be accomplished,for example, by utilizing mixed functionality adsorbents. Once theresolution of the analyte is accomplished with relatively lowspecificity, the binding characteristic found to attract the analyte ofinterest can be exploited in combination with a variety of other bindingand elution characteristics to remove still more undesired componentsand thereby resolve the analyte.

For example, if the analyte binds to hydrophobic adsorbents, the analytecan be further resolved from other hydrophobic sample components byproviding a mixed functionality adsorbent which exhibits as one basis ofattraction a hydrophobic interaction and also exhibits a second,different basis of attraction. The mixed functionality adsorbent mayexhibit hydrophobic interactions and negatively charged ionicinteractions so as to bind hydrophobic analytes which are positivelycharged. Alternatively, the mixed functionality adsorbent can exhibithydrophobic interactions and the ability to form coordinate covalentbonds with metal ions so as to bind hydrophobic analytes having theability to form coordination complexes with metal ions on the adsorbent.Still further examples of adsorbents exhibiting binding characteristicsof intermediate specificity will be readily apparent to those skilled inthe art based upon the disclosure and examples set forth above.

The resolution of analytes on the basis of binding characteristics ofintermediate specificity can be further by exploiting bindingcharacteristics of relatively high specificity. Binding characteristicsof relatively high specificity can be exploited by utilizing a varietyof adsorbents exhibiting the same basis of attraction but a differentstrength of attraction. In other words, although the basis of attractionis the same, further resolution of the analyte from other samplecomponents can be achieved by utilizing adsorbents having differentdegrees of affinity for the analyte.

For example, an analyte that binds an adsorbent based upon the analyte'sacidic nature may be further resolved from other acidic samplecomponents by utilizing adsorbents having affinity for analytes inspecific acidic pH ranges. Thus the analyte may be resolved using oneadsorbent attracted to sample components of pH 1-2, another adsorbentattracted to sample components of pH of 3-4, and a third adsorbentattracted to sample components of pH of 5-6. In this manner, an analytehaving a specific affinity for an adsorbent which binds analyte of pH of5-6 will be resolved from sample components of pH of 1-4. Adsorbents ofincreasing specificity can be utilized by decreasing the interval ofattraction, i.e., the difference between the binding characteristics ofadsorbents exhibiting the same basis of attraction.

A primary analyte adsorbed to a primary adsorbent can, itself, haveadsorbent properties. In this case, the primary analyte and adsorbed toa substrate can become a secondary absorbent for isolating secondaryanalytes. In turn, the retained secondary analyte can function as atertiary adsorbent to isolate a tertiary analyte from a sample. Thisprocess can continue through several iterations.

2. Eluants

The eluants, or wash solutions, selectively modify the threshold ofadsorption between the analyte and the adsorbent. The ability of aneluant to desorb and elute a bound analyte is a function of its elutioncharacteristics. Different eluants can exhibit grossly different elutioncharacteristics, somewhat different elution characteristics, or subtlydifferent elution characteristics.

The temperature at which the eluant is contacted to the adsorbent is afunction of the particular sample and adsorbents selected. Typically,the eluant is contacted to the adsorbent at a temperature of between 0°C. and 100° C., preferably between 4° C. and 37° C. However, for someeluants, modified temperatures can be desirable and will be readilydeterminable by those skilled in the art.

As in the case of adsorbents, eluants which exhibit grossly differentelution characteristics generally differ in their basis of attraction.For example, various bases of attraction between the eluant and theanalyte include charge or pH, ionic strength, water structure,concentrations of specific competitive binding reagents, surfacetension, dielectric constant and combinations of two or more of theabove.

a. pH-Based Eluants

Eluants which modify the selectivity of the adsorbent based upon pH(i.e., charge) include known pH buffers, acidic solutions, and basicsolutions. By washing an analyte bound to a given adsorbent with aparticular pH buffer, the charge can be modified and therefore thestrength of the bond between the adsorbent and the analyte in thepresence of the particular pH buffer can be challenged. Those analyteswhich are less competitive than others for the adsorbent at the pH ofthe eluant will be desorbed from the adsorbent and eluted, leaving boundonly those analytes which bind more strongly to the adsorbent at the pHof the eluant.

b. Ionic Strength-Based Eluants

Eluants which modify the selectivity of the adsorbent with respect toionic strength include salt solutions of various types andconcentrations. The amount of salt solubilized in the eluant solutionaffects the ionic strength of the eluant and modifies the adsorbentbinding ability correspondingly. Eluants containing a low concentrationof salt provide a slight modification of the adsorbent binding abilitywith respect to ionic strength. Eluants containing a high concentrationof salt provide a greater modification of the adsorbent binding abilitywith respect to ionic strength.

c. Water Structure-Based Eluants

Eluants which modify the selectivity of the adsorbent by alteration ofwater structure or concentration include urea and chaotropic saltsolutions. Typically, urea solutions include, e.g., solutions ranging inconcentration from 0.1 to 8 M. Chaotropic salts which can be used toprovide eluants include sodium thiocyanate. Water structure-basedeluants modify the ability of the adsorbent to bind the analyte due toalterations in hydration or bound water structure. Eluants of this typeinclude for example, glycerol, ethylene glycol and organic solvents.Chaotropic anions increase the water solubility of nonpolar moietiesthereby decreasing hydrophobic interactions between the analyte and theadsorbent.

d. Detergent-Based Eluants

Eluants which modify the selectivity of the adsorbent with respect tosurface tension and analyte structure include detergents andsurfactants. Suitable detergents for use as eluants include ionic andnonionic detergents such as CHAPS™ (3-[3-(cholamidopropyl)dimethylammonio]-1-propane-sulfonate), TWEEN™ and Tergitol NP-40 (nonylphenoxypolyethoxy ethanol). Detergent-based eluants modify the ability of theadsorbent to bind the analyte as the hydrophobic interactions aremodified when the hydrophobic and hydrophilic groups of the detergentare introduced. Hydrophobic interactions between the analyte and theadsorbent, and within the analyte are modified and charge groups areintroduced, e.g., protein denaturation with ionic detergents such as SDS(sodium dodecyl sulfate).

c. Hydrophobicity-Based Eluants

Eluants which modify the selectivity of the adsorbent with respect todieelectric constant are those eluants which modify the selectivity ofthe adsorbent with respect to hydrophobic interaction. Examples ofsuitable eluants which function in this capacity include urea (0.1-8M)organic solvents such as propanol, acetonitrile, ethylene glycol andglycerol, and detergents such as those mentioned above. Use ofacetonitrile as eluant is typical in reverse phage chromatography.Inclusion of ethylene glycol in the eluant is effective in elutingimmunoglobulins from salt-promoted interactions with thiophilicadsorbents.

f. Combinations of Eluants

Suitable eluants can be selected from any of the foregoing categories orcan be combinations of two or more of the foregoing eluants. Eluantswhich comprise two or more of the foregoing eluants are capable ofmodifying the selectivity of the adsorbent for the analyte on the basisof multiple elution characteristics.

3. Variability of Two Parameters

The ability to provide different binding characteristics by selectingdifferent adsorbents and the ability to provide different elutioncharacteristics by washing with different eluants permits variance oftwo distinct parameters each of which is capable of individuallyeffecting the selectivity with which analytes are bound to theadsorbent. The fact that these two parameters can be varied widelyassures a broad range of binding attraction and elution conditions sothat the methods of the present invention can be useful for binding andthus detecting many different types of analytes.

The selection of adsorbents and eluants for use in analyzing aparticular sample will depend on the nature of the sample, and theparticular analyte or class of analytes to be characterized, even if thenature of the analytes are not known. Typically, it is advantageous toprovide a system exhibiting a wide variety of binding characteristicsand a wide variety of elution characteristics, particularly when thecomposition of the sample to be analyzed is unknown. By providing asystem exhibiting broad ranges of selectivity characteristics, thelikelihood that the analyte of interest will be retained by one or moreof the adsorbents is significantly increased.

One skilled in the art of chemical or biochemical analysis is capable ofdetermining the selectivity conditions useful for retaining a particularanalyte by providing a system exhibiting a broad range of binding andelution characteristics and observing binding and elutioncharacteristics which provide the best resolution of the analyte.Because the present invention provides for systems including broadranges of selectivity conditions, the determination by one skilled inthe art of the optimum binding and elution characteristics for a givenanalyte can be easily accomplished without the need for undueexperimentation.

C. Analytes

The present invention permits the resolution of analytes based upon avariety of biological, chemical, or physico-chemical properties of theanalyte by exploiting the properties of the analyte through the use ofappropriate selectivity conditions. Among the many properties ofanalytes which can be exploited through the use of appropriateselectivity conditions are the hydrophobic index (or measure ofhydrophobic residues in the analyte), the isoelectric point (i.e., thepH at which the analyte has no charge), the hydrophobic moment (ormeasure of amphipathicity of an analyte or the extent of asymmetry inthe distribution of polar and nonpolar residues), the lateral dipolemoment (or measure of asymmetry in the distribution of charge in theanalyte), a molecular structure factor (accounting for the variation insurface contour of the analyte molecule such as the distribution ofbulky side chains along the backbone of the molecule), secondarystructure components (e.g., helix, parallel and antiparallel sheets),disulfide bands, solvent-exposed electron donor groups (e.g., His),aromaticity (or measure of pi-pi interaction among aromatic residues inthe analyte) and the linear distance between charged atoms.

These are representative examples of the types of properties which canbe exploited for the resolution of a given analyte from a sample by theselection of appropriate selectivity characteristics in the methods ofthe present invention. Other suitable properties of analytes which canform the basis for resolution of a particular analyte from the samplewill be readily known and/or determinable by those skilled in the artand are contemplated by the instant invention.

The invention method is not limited with respect to the types of sampleswhich can be analyzed. Samples can be in the solid, liquid, or gaseousstate, although typically the sample will be in a liquid state. Solid orgaseous samples are preferably solubilized in a suitable solvent toprovide a liquid sample according to techniques well within the skill ofthose in the art. The sample can be a biological composition,non-biological organic composition, or inorganic composition. Thetechnique of the present invention is particularly useful for resolvinganalytes in a biological sample, particularly biological fluids andextracts; and for resolving analytes in non-biological organiccompositions, particularly compositions of small organic and inorganicmolecules.

The analytes may be molecules, multimeric molecular complexes,macromolecular assemblies, cells, subcelluar organelles, viruses,molecular fragments, ions, or atoms. The analyte can be a singlecomponent of the sample or a class of structurally, chemically,biologically, or functionally related components having one or morecharacteristics (e.g., molecular weight, isoelectric point, ioniccharge, hydrophobic/hydrophilic interaction, etc.) in common.

Specific examples of analytes which may be resolved using the retentatechromatography methods of the present invention include biologicalmacromolecules such as peptides, proteins, enzymes, polynucleotides,oligonucleotides, nucleic acids, carbohydrates, oligosaccharides,polysaccharides; fragments of biological macromolecules set forth above,such as nucleic acid fragments, peptide fragments, and proteinfragments; complexes of biological macromolecules set forth above, suchas nucleic acid complexes, protein-DNA complexes, receptor-ligandcomplexes, enzyme-substrate, enzyme inhibitors, peptide complexes,protein complexes, carbohydrate complexes, and polysaccharide complexes;small biological molecules such as amino acids, nucleotides,nucleosides, sugars, steroids, lipids, metal ions, drugs, hormones,amides, amines, carboxylic acids, vitamin and coenzymes, alcohols,aldehydes, ketones, fatty acids, porphyrins, carotenoids, plant growthregulators, phosphate esters and nucleoside diphosph-sugars, syntheticsmall molecules such as pharmaceutically or therapeutically effectivesugars, monomers, peptide analogs, steroid analogs, inhibitors,mutagens, carcinogens, antimitotic drugs, antibiotics, ionophores,antimetabolites, amino acid analogs, antibacterial agents, transportinhibitors, surface-active agents (surfactants), mitochondrial andchloroplast function inhibitors, electron donors, carriers andacceptors, synthetic substrates for proteases, substrates forphosphatases, substrates for esterases and lipases and proteinmodification reagents; and synthetic polymers, oligomers, and copolymerssuch as polyalylenes, polyamides, poly(meth)acrylates, polysulfones,polystyrenes, polyethers, polyvinyl ethers, polyvinyl esters,polycarbonates, polyvinyl halides, polysiloxanes, POMA, PEG, andcopolymers of any two or more of the above.

III. Information Processing

Detection of analytes adsorbed to an adsorbent under particular elutionconditions provides information about analytes in a sample and theirchemical character. Adsorption depends, in part, upon the bindingcharacteristics of the adsorbent: Analytes that bind to an adsorbentpossess the characteristics that makes binding possible. For example,molecules that are cationic at a particular pH will bind to an anionicadsorbent under elution conditions that include that pH. Stronglycationic molecules will only be eluted from the adsorbent under verystrong elution conditions. Molecules with hydrophobic regions will bindto hydrophobic adsorbents, while molecules with hydrophilic regions willbind to hydrophilic adsorbents. Again, the strength of the interactionwill depend, in part, upon extreme to which an analyte containshydrophobic or hydrophilic regions. Thus, the determination that certainanalytes in a sample bind to an adsorbent under certain elutionconditions not only resolves analytes in a mixture by separating themfrom each other and from analytes that do not possess the appropriatechemical character for binding, but also identifies a class of analytesor individual analytes having the particular chemical character.Collecting information about analyte retention on one or more particularadsorbents under a variety of elution conditions provides not onlydetailed resolution of analytes in a mixture, but also chemicalinformation about the analytes, themselves that can lead to theiridentity. This data is referred to as “retention data.”

Data generated in retention assays is most easily analyzed with the useof a programmable digital computer. The computer program generallycontains a readable medium that stores codes. Certain code is devoted tomemory that includes the location of each feature on a substrate array,the identity of the adsorbent at that feature and the elution conditionsused to wash the adsorbent. Using this information, the program can thenidentify the set of features on the array defining certain selectivitycharacteristics. The computer also contains code that receives as input,data on the strength of the signal at various molecular masses receivedfrom a particular addressable location on the probe. This data canindicate the number of analytes detected, optionally including for eachanalyte detected the strength of the signal and the determined molecularmass.

The computer also contains code that possess the data. This inventioncontemplates a variety of methods for processing the data. In oneembodiment, this involves creating an analyte recognition profile. Forexample, data on the retention of a particular analyte identified bymolecular mass can be sorted according to a particular bindingcharacteristics, for example, binding to anionic adsorbents orhydrophobic adsorbents. This collected data provides a profile of thechemical properties of the particular analyte. Retention characteristicsreflect analyte function which, in turn, reflects structure. Forexample, retention to coordinate covalent metal chelators can reflectthe presence of histidine residues in a polypeptide analyte. Using dataof the level of retention to a plurality of cationic and anionicadsorbents under elution at a variety of pH levels reveals informationfrom which one can derive the isoelectric point of a protein. This, inturn, reflects the probable number of ionic amino acids in the protein.Accordingly, the computer can include code that transforms the bindinginformation into structural information. Furthermore, secondaryprocessing of the analyte (e.g., post-translational modifications)results in an altered recognition profile reflected by differences inbinding or mass.

In another embodiment, retention assays are performed under the same setof selectivity thresholds on two different cell types, and the retentiondata from the two assays is compared. Differences in the retention maps(e.g., presence or strength of signal at any feature) indicate analytesthat are differentially expressed by the two cells. This can include,for example, generating a difference map indicating the difference insignal strength between two retention assays, thereby indicating whichanalytes are increasingly or decreasingly retained by the adsorbent inthe two assays.

The computer program also can include code that receives instructionsfrom a programmer as input. The progressive and logical pathway forselective desorption of analytes from specified, predetermined locationsin the array can be anticipated and programmed in advance.

The computer can transform the data into another format forpresentation. Data analysis can include he steps of determining, e.g.,signal strength as a function of feature position from the datacollected, removing “outliers” (data deviating from a predeterminedstatistical distribution), and calculating the relative binding affinityof the analytes from the remaining data.

The resulting data can be displayed in a variety of formats. In oneformat, the strength of a signal is displayed on a graph as a functionof molecular mass. In another format, referred to as “gel format,” thestrength of a signal is displayed along a linear axis intensity ofdarkness, resulting in an appearance similar to bands on a gel. Inanother format, signals reaching a certain threshold are presented asvertical lines or bars on a horizontal axis representing molecular mass.Accordingly, each bar represents an analyte detected. Data also can bepresented in graphs of signal strength for an analyte grouped accordingto binding characteristics and/or elution characteristics.

IV. Applications of Retentate Chromatography

Retentate chromatography involves a combinatorial separation method,including detection and characterization of multiple analytes inparallel. These combinatorial methods have many applications. Suchapplications include, without limitation, developing target analytedetection schemes; developing protein purification strategies; proteinpurification methods; identifying specific phage from a phage displaylibrary that bind to a target analyte, including target epitopeidentification using complementary phage display libraries; proteinidentification based on physico-chemical properties of the analyte; geneexpression monitoring and differential protein display; toxicologyscreening; simultaneous detection of multiple diagnostic markers; drugdiscovery; multimeric protein assembly monitoring and detection of invitro polynucleotide translation.

A. Methods for Sequentially Extracting Analytes from a Sample

Retentate chromatography involves the analysis of retention of ananalyte under a plurality of adsorbent/eluent conditions. One variationof this method is sequential extraction. In sequential extraction asample is not independently exposed to two different selectivityconditions. Rather, the sample is exposed to a first selectivitycondition to extract certain analytes from the sample onto theadsorbent, and leave non-adsorbed analytes in the eluent. Then, theeluent is exposed to a second selectivity condition. This furtherextracts various analytes from the eluant. Frequently, if the adsorbentsin the first and second exposure have different basis for attraction(e.g., normal phase and hydrophobic) the adsorbent will extract adifferent set of analytes from the eluent. This second eluant is thenexposed to a third selectivity condition, and so on. In one method ofpracticing sequential extraction, the adsorbent is placed at the bottomof a well so that sample can be mixed on top of it. An eluant is addedto the adsorbent and after allowing binding between analytes in thesample, the eluant wash is collected. The collected wash is then exposedto a second adsorbent, and analytes are extracted from the sample bybinding.

In one embodiment, the goal of sequential extraction is preparativerather than analytical. More specifically, the goal may be to extractall but a desired analyte from the sample. In this case, the sample isusually small, e.g., a few microliters on a spot about a few millimetersin diameter. The adsorbents are selected so as not to adsorb an analyteone wishes not to be depleted from the sample. After several iterationsthe finally collected wash is depleted of un-desired analytes, leavingthe desired ones for subsequent analysis by, for example, desorptionspectrometry or traditional chromatographic methods.

In another embodiment, unretained sample is, itself, analyzed oranalytes by any analytical technique. Even after a single retentionstep, this process allows one to examine materials adsorbed to anadsorbent and those analytes that are not adsorbed.

B. Methods for Progressive Resolution of Analytes in a Sample

One object of retentate chromatography is the unambiguous resolution ofan analyte from a complex sample mixture. This is especially importantfor applications in clinical diagnostics, drug discovery and functionalgenomics. These areas can involve the identification of one or moreanalytes from a biological sample. This invention provides a method foridentifying selectivity conditions with improved resolution for ananalyte. The method involves identifying a selectivity condition inwhich the analyte is retained and, in an iterative process, addingadditional binding characteristics or elution characteristics to theselectivity condition which provide improved resolution of the analyte.

A mass spectrum of a complex sample exposed to a selectivity conditiongenerally includes signals from many components of the sample. Thecomplexity of the signals may interfere with unambiguous resolution ofthe analyte. Methods for progressive resolution of an analyte allow oneto identify selectivity conditions with improved resolution of theanalyte for unambiguous detection of an analyte in a sample. Aselectivity condition exhibits “improved resolution” of an analytecompared with another selectivity condition if the analyte signal ismore easily distinguishable from the signals of other components. Thiscan include, for example, decreasing the number of analytes bound to theadsorbent, thereby decreasing the total number of signals, or increasingthe selectivity of the selectivity condition for the analyte, therebyenhancing the analyte signal compared with other signals. Of course,when the analyte is exclusively bound to the substrate, it generates thesole analyte signal during detection.

Methods of progressive resolution involve an iterative process in whichadditional selectivity (binding or elution) characteristics aresequentially added to a constant set of selectivity characteristicsknown to retain the analyte. In a first step a series of selectivityconditions are tested to identify one that retains the target analyte.In a next step, one or more of the selectivity characteristics of theselectivity condition are selected for the constant set for furtheranalysis.

A new set of selectivity conditions is generated. Each of the conditionsin the new set includes the selected characteristics in the constantset, and at least one new condition not in the constant set. Forexample, if the constant set includes an anionic adsorbent and a lowsalt eluant, the new condition could involve varying the pH of theeluant. Each of these new variables is tested for the ability to improvethe resolution of the analyte, and one modified selectivity conditionwith improved resolution is identified. In a next step, an addedselectivity condition that provides improved resolution is added to theconstant set.

The modified constant set is tested again in the same way, by generatinga new set of selectivity conditions that include the characteristics ofthe constant set and a et of new characteristics. Thus, at each step,the selectivity conditions are selected so that resolution of theanalyte is improved compared with the selectivity condition at aprevious step.

The method is well described by example. A cell sample typicallycontains hundreds or perhaps thousands of proteins. One may wish toobtain unambiguous resolution of a single target protein analyte in thesample. In a first step, a retention map is developed for the targetanalyte using a plurality of selectivity conditions. For example, theadsorbent could be an anion exchanger, a cation exchanger, a normalphase adsorbent and a revere phase adsorbent. The elution conditionstested on each adsorbent could be a variety of pH levels, a variety ofionic strengths, a variety of detergent conditions and a variety ofhydrophobicity-based conditions. For example, four different elutionconditions could be tested for each condition. Thus, in this example,sixteen different selectivity conditions are tested for their ability toadsorb the target analyte.

From this retention map one selects at least one selectivity conditionunder which the target analyte is retained. One may select a selectivitycondition under which the target bound maximally. However, it may beadvantageous to select a condition under which the target is notmaximally bound if this selectivity condition is more selective for thetarget than the other selectivity conditions. Presume, for this example,that analysis of the retention map shows that the target is retained byanion exchange adsorbents at around neutral pH, but also is weaklyadsorbed to a hydrophilic adsorbent.

One variable absorbent or eluant from the selectivity conditionidentified to result in retention of the analyte is then selected foruse on all subsequent selectivity conditions. As used herein, it is saidto be added to the “set of selectivity condition constants.”

In the next iteration, one tests the ability of the target analyte tobind under a second plurality of selectivity conditions. Eachselectivity condition at the second set includes the elements of theselectivity condition constant set. However, each selectivity alsoincludes another variable—a different adsorbent or eluant added to theselectivity condition. Thus, within the constraint of employing at leastthe set of constants, the second set of selectivity conditions also arechosen to be more diverse than the first set. Methods of increasing thediversity include, for example, testing finer gradations of an elutioncondition or different strengths of an adsorbent. It also can include,for example, the addition of another selectivity characteristic into theselectivity conditions.

Continuing the example, the anion exchange adsorbent may be added to theset of constants. This condition is now tested with a wider variety ofvariables, e.g. eluants or adsorbents. Eluants to be tested can includea variety of low ph buffers at finer gradations than tested in the firstiteration. For example, the first iteration may have tested buffers atpH 3.0, pH 5.0, pH 7.0 and pH 9.0, and showed that the target bound tothe anion exchange adsorbent near neutral pHs. During the seconditeration, the buffers tested could be at pH 5.0, pH 5.5, pH 6.0, pH6.5, pH 7.0, pH 7.5 and pH 8.0. In addition, each of these buffers alsocould be varied to include other elution characteristics, e.g., ionicstrength, hydrophobicity, etc.

Analysis of the second retention map resulting at this stage generallywill allow one to identify a condition that provided better resolutionthan the selectivity condition identified in the first round. Again, oneof the variables of this selectivity condition is chosen and added tothe set of selectivity condition constants for further interrogation inthe next interation.

Continuing the example, suppose the selectivity condition in the secondround that resolves the analyte best uses a buffer at pH 6.5. Thiselutan can now be added to the set of constants, which now includes ananion exchange resin and a pH 6.5 buffer. In the next iteration, theselectivity conditions include this constant set, and another variable.The variable might be, for example, addition of a new component to theeluant, such as different ionic strengths; or another adsorbent can beadded into the mixture, such as variety of hydrophobic adsorbents mixedwith the anion exchange adsorbent; or one may vary the density of theanion exchange resin. Again, a selectivity condition is identified fromthis set that shows improved resolution of the analyte.

The process can continue until the analyte is purified to essentialhomogeneity. In this case, the selectivity condition is specific for theanalyte.

As one can see, by increasing the number of variables tested at eachstep, one can decrease the number of iteration needed to identify asuitable selectivity condition.

C. Methods Of Preparative Purification Of An Analyte

In another aspect, this invention provides methods of purifying ananalyte. The methods take advantage of the power of retentatechromatography to rapidly identify bases of attraction for adsorbing ananalyte. A first step involve exposing the analyte to a plurality ofselectivity conditions and determining retention under the conditions byretentate chromatography. This generates a recognition profilecharacteristics of the analyte. The selectivity conditions under whichthe analyte is retained are used to develop a protocol for preparativepurification of the analyte.

For preparative purification of the analyte, the analyte is sequentiallyadsorbed and eluted from a series of adsorbent/eluant combinations thatwere identified as binding the analyte. Thus, for example, therecognition map may indicate that the analyte binds to a normal phaseadsorbent and to a metal chelator. The analyte is then contacted with afirst chromatography column, for example, containing the normal phaseabsorbent, which binds the analyte. Unbound material is washed off. Thenthe analyte is eluted by a sufficiently stringent wash. The eluant isthen contacted with a metal chelate column, for example, to bind theanalyte. Unbound material are washed away. Then, the bound material thatincludes the analyte, is eluted from the metal chelate column. In thisway, the analyte is isolated in preparation amounts. A preparativeamount of a sample is at least 10 μl, at least 100 μl, at least 1 ml orat least 10 ml.

The information generated during progressive resolution of analytes canbe used to deign larger scale chromatographic (elution-based) proteinpurification strategies. The adsorbent bases for attraction, the bindingconditions, and the elution conditions (i.e., the selectivity condition)for a target analyte protein become defined by retentate chromatography.This information can save an enormous amount of time, energy, andprecious analyte that would otherwise be washed during the trial anderror process of purification strategy design that is now in place. Thissection also provides for large scale purification efforts performedwith commercially available adsorbents.

D. Methods For Making Probes For Specific Detection Of Analytes

This invention provides probes for the specific detection of one or moreanalytes by desorption spectrometry, as well as methods for generatingthese probes. Such analytes-resolving probes are useful the specificdetection of analytes in diagnostic and analytic methods.

The first step in generating a probe for resolving one or more analytesis to produce a retention map for the analytes under a plurality ofdifferent adsorbent/eluant combinations. For example, the resolution ofthe analytes can be determined for four different adsorbents washed witheach of five different eluants. This provides twenty sets of retentiondata for each of the analytes. Analysis of the resulting retention mapwill indicate which selectivity condition or conditions best resolvesthe analytes. Preferably, one selectivity condition can be identifiedthat unambiguously resolves all the analytes. Then, one or moreselectivity conditions is selected for use in the analyte-resolvingprobe so that each of the analytes is resolved on at least one adsorbentspot. The probe also could contain an adsorbent that does not bind theanalyte or analytes. This adsorbent spot is useful as a control. Theprobes can include a plurality of adsorbent spots in addressablelocations selected for their ability to retain and resolve the analyteor analyte. In this case, adsorbents are selected that bind the analyteunder a single eluant condition. This is useful because the entire probecan be washed with a single eluant in the detection process.

The retention map generated for a particular analyte can be used createa customized adsorbent for the analyte. For example, the nature of theadsorbents that retain an analyte indicate a set of bases for attractionof an analyte. A customized adsorbent can be designed by preparing amultiplex adsorbent that includes elements of adsorbents that providethese bases for attraction. Such a custom adsorbent is very selectivefor the target analyte. One or a few custom adsorbents can suffice togenerate a recognition map for the analyte. For example, if it is foundthat under particular elution conditions an analyte is retained byadsorbents that bind materials that have certain degree ofhydrophobicity, positive charge and aromaticity, one can create a customadsorbent by design or through the use of combinatorial syntheticstrategies having functional groups that attract each of thesecharacteristics. Detecting binding to this adsorbent identifies theanalyte.

Such probes are useful for detecting the analyte or analytes in asample. The sample is exposed to the selectivity conditions and theprobe is interrogated by desorption spectrometry. Because the proberesolves the analytes, their presence can be detected by looking for thecharacteristic recognition profile. Such probes are particularly usefulfor identifying a set of diagnostic markers in a patient sample.

In one embodiment, the array is designed to dock specific classes ofprotein of interest. This includes diagnostic markers as well asanalytes defined by function. For example, an array can be prepared thatspecifically docks cell surface protein, enzymes of a certain class(e.g., kinases), transcription factor, intracellular receptors, etc. Theadsorbent can be specific for the biopolymers, for example, antibodies.

In one embodiment, the adsorbents are genetic packages such as phagedisplaying protein ligands for a certain class of protein. In this case,a phage display library can be pre-screened with a certain class ofmolecules to eliminate phage that bind too that class. Then, phage thathave been subtracted from the population are used as adsorbents.

E. Diagnostic Probes And Methods Of Diagnosis

Diagnosis of pathological conditions frequently involves the detector ina patient sample of one or more molecular markers or disease. Certainconditions can be diagnosed by the presence of a single diagnosticmarker. Diagnosis of other conditions may involve detection of aplurality diagnostic markers. Furthermore, the detection of severalmarkers may increase the confidence of diagnosis. Accordingly, thisinvention provides probes for desorption spectrometry comprising atleast one adsorbent that resolves at least one diagnostic marker of apathological condition.

The preparation of such probes involves, first, the selection of markersto be detected. The marker can be a marker for any disease state, e.g.,cancer, heart disease, autoimmune disease, viral infection, Alzheimer'sdisease or diabetes. For example, detection of prostate specific antigen(PSA) is highly suggestive or prostate cancer. HIV infection can bediagnosed by detecting antibodies against several HIV proteins, such asone of p17, p24 or p55 and one of p31, p51 or p66 and one of gp41 orgp120/160. Detection of amyloid-β42 and tau protein in cerebrospinalfluid is highly indicative of Alzheimer's disease. Also, the markers canbe identified by methods of this invention involving detectingdifferential presence of an analyte in healthy subjects versus subjectswith pathological conditions.

In a next step, adsorbents are developed that retain one or morediagnostic markers. Preferably, a single adsorbent is prepared thatresolves all the markers. This can be accomplished, for example, bycreating a spot containing several antibodies, each of which binds oneof the desired markers. Alternatively, the probe can comprise aplurality of adsorbent spots, each spot capable of resolving at leastone target analyte under a selectivity condition. In one embodiment, theadsorbent is a multiplex adsorbent comprising ligands that are specificfor the markers. For example, the adsorbent can comprise an antibody, apolypeptide ligand or a polynucleotide that specifically binds thetarget analyte. In one embodiment, the antibody is a single chainantibody identified by screening a combinatorial library. Single chainantibodies that are specific for particular markers can be developed byscreening phage display libraries by methods described herein.

In another embodiment, the adsorbent comprises non-organic biomolecularcomponents that either retain the target analyte specifically or thatretain the analyte with sufficient specificity for unambiguousresolution by desorption spectrometry. Preparation of adsorbents fordetection of specific analyte also are described herein.

Significantly, a single adsorbent spot used in these methods need not bespecific for a single analyte and, therefore, need not requirebiopolymer-mediated specific affinity between target and adsorbent.Prior affinity detection methods have relied mainly on specific bindingbetween a biopolymer and a target. This includes, for example, thespecific affinity of an antibody for a protein, a polynucleotide for acomplementary polynucleotide or a lectin for a carbohydrate. Suchspecificity was necessary because these means of detection wereindirect: the target was not identified; a label, frequently bound tothe adsorbent, was identified. Accordingly, the more specific theadsorbent, the less likelihood that contaminants would bind to theadsorbent and interfere with specific detection. However, desorptionspectrometry results in direct detection of an analyte. Accordingly, thepresence of contaminants does not interfere with specific detectionunless the signal of the contaminant overlaps with the signal of thetarget.

Methods of diagnosis involve, first, selecting a patient sample to betested. The sample can be, e.g., tissue, blood, urine, stool or otherbodily fluid (lymph, cerebrospinal, interarticular, etc.). Then, thesample is exposed to a substrate containing the diagnostic adsorbentsunder conditions to allow retention of the diagnostic markers. Theadsorbent is washed with an appropriate eluant. Then the markers aredetected (e.g., resolved) by desorption spectrometry (e.g., massspectrometry).

This invention also provides kits for specific detection of diagnosticmarkers including (1) a substrate for use in desorption spectrometrythat comprises at least one adsorbent in at least one addressablelocation that resolves at least one diagnostic marker under aselectivity condition that comprises the adsorbent and an eluant and (2)the eluant or instructions for preparation of the eluant. Upon exposingthe sample to the adsorbent and washing with the eluant, i.e., byexecuting the selectivity condition, the analyte is sufficientlypurified or specifically bound for resolution by desorptionspectrometry.

F. Methods For Identifying Proteins

In another aspect, this invention provides a method for aiding in theidentification of a protein. The method involves determining matchparameters for physico-chemical characteristics of a protein analyteusing retentate chromatography and searching a protein database toidentify proteins having the match parameters. The derivation ofphysico-chemical information based on retention characteristics isdiscussed above. The database typically will provide the amino acidsequence and/or the nucleotide sequence encoding the amino acid sequenceof each protein. Structural characteristics, such as molecular mass,hydrophobicity, pI, fragment mass, etc. are easily derivable from thisinformation. An analyte protein will share any particular structuralcharacteristic with only a subset of the protein in the database.Accordingly, identity candidate are found by sorting the proteinsaccording to structural characteristics shared with the protein analyte.Thus, in view of the inaccuracy, degree of specificity, or level ofconfidence inherent in identifying one or more physicochemicalproperties of the reference, one cannot except that proteins in thedatabase will perfectly match all the characteristics of the reference.Accordingly, the match parameters can be set to identify, for example,the closeness of fit between the protein analyte characteristics and thecharacteristic of the reference polypeptides in the database.

As our identification of genes in the genome increases, the chance thatany protein analyte exists in the database as a reference polypeptidealso increases. Accordingly, this method enables one to rapidly resolvea protein of interest in a sample, obtain structural information aboutthe protein, and then use this information to identify the protein.

G. Methods For Assembling Multimeric Molecules

The ability of adsorbents to dock desired molecules is useful inbuilding multimeric molecules and assessing compounds that effect theirassembly. A unit of the multimeric molecule is bound to an absorber.Then it is exposed to a sample that contains another unit of themultimeric molecule. Expose can be performed under a variety ofconditions to test binding parameters. The binding of a subunit to themultimer can be monitored by desorption spectrometry. Then, a subsequentsubunit can be tested for binding in the same say. The drug screeningmethods described herein are useful for testing agent for the ability tointerfere with assembly. Accordingly, an analyte at one stage of theprocess becomes an adsorbent at the next stage.

H. Methods For Performing Enzyme Assays

This invention also provides methods for performing enzyme assays.Enzyme assays generally involve exposing a sample to be tested with anenzyme substrate under conditions used under which the enzyme is active.After allowing the enzyme to act on the substrate, a product of theenzymatic reaction is detected. In quantitative assays, the amount ofproduct is determined. This amount usually is compared to a control or astandard curve, thereby yielding an amount of enzyme activity in thesample.

The invention provides methods for detecting an enzyme, includingdetecting an amount of enzyme activity, in a sample. The method takesadvantage of the fact that the activity of an enzyme often produces aproduct whose mass is different than the original substrate. In themethod, a solid phase is prepared that comprises an adsorbent that bindsthe substrate. An amount of the substrate i bound to the adsorbent. Thenthe adsorbent is exposed to the sample under conditions and for a timethat allows any enzyme to act on the substrate. Then, any bound materialis detected by desorption spectrometry. Detection of an analyte having amolecular mass characteristic of the product of enzyme activity providean indication of the presence of the enzyme. The signal strength will bea function of the amount of enzymatic activity in the sample.

1. Method For Identifying Analytes That Are Differentially ExpressedBetween Biological Materials

In another aspect this invention provides methods for identifyingorganic biomolecules, particularly proteins, that are differentiallyexpressed between two or more samples. “Differential expression” referto differences in the quantity of quality of an analyte between twosamples. Such differences could result in any stage of proteinexpression from transcription through post-translation modification. Themethods are advantage of the extraordinary resoling power andsensitivity or retentate chromatography. First, recognition profilesusing the same set of selectivity conditions are prepared for analytefrom the two biological samples. The greater the number of selectivityconditions used, the greater the resolution of analytes in the sampleand, therefore, the greater the number of analytes that can be compared.Then, the recognition maps are compared to identify analyte that aredifferentially retained by the two sets of adsorbents. Differentialretention includes quantitative retention. This indicates, e.g., up- ordown-regulation of expression. Differential retention also includesqualitative differences in the analyte. For example, differences inpost-translational modification of a protein can result in differencesin recognition maps detectable as differences in binding characteristics(for example, if the protein is glycosylated, it will bind differentlyto lectin adsorbents) or differences in mass (for example, as a resultof differences in post-translational cleavage). The analysis can becarried out by a programmable, digital computer.

The method is particularly useful to detect genes that aredifferentially expressed between two cell types. The two cell typescould be normal versus pathologic cells, e.g., cancer cells or cell atdifferent levels or cells at different stages of development ordifferentiation, or in different parts of the cell cycle. However, themethod also is useful in examining two cells of the same type exposed todifferent conditions. For example, the method is useful in toxicologyscreening and testing agents for the ability to modulate gene expressionin a cell. In such a method, one biological sample is exposed to thetest agent, and other cell is not. Then, retentate maps of the sampleare compared. This method may indicate that a protein or otherbiomolecule is increased or decreased in expression, or is changed someway based on different retention characteristics or different mass.

Using information about the physico-chemical properties ofdifferentially expressed proteins obtained from the retention maps,identity candidates for these proteins can be determined using methodsdescribed herein.

This method is useful for identifying diagnostic markers of disease.Proteins that are differentially expressed in a patient sample or adiseased cultured cell compared to normal samples or cells may bediagnostic markers. In general, it is best to compare samples from astatistically significant patient population with normal samples. Inthis way, information can be pooled to identify diagnostic markerscommon to all or a significant number of individuals exhibiting thepathology.

1. Increasing Sensitivity by Catabolic Signal Amplification

The sensitivity of detecting differential presence (e.g., resulting fromdifferential expression) of large proteins in a complex mixture can beincreased significantly by fragmenting the large protein into smallerpieces and detecting the smaller pieces. Increased sensitivity is due toseveral factors. First, when all the proteins in a sample are fragmentedby, for example, enzymatic digestion, large proteins are likely toproduce more fragments than small proteins. Second, the overallsensitivity of deposition spectrometry is greater at lower molecularmasses than higher molecular masses. Third, fragmenting a proteinincreases the number of signals from that target, thereby increasing thelikelihood of detecting the target. Fourth, fragmenting a proteinincreases the likelihood of capturing and, therefore, detecting, atleast one fragment of the protein. Fifth, if a protein is differentiallypresent in two samples, then by increasing the number of signals fromthat protein, the difference in amount is more likely to be detected.

Also, the method is counter-intuitive. Generally, one seeks to decreasethe complexity of an analyte mixture before analysis. Fragmentationincreases the complexity.

Accordingly, in one embodiment of this invention the sensitivity ofdetecting an analyte is increased by converting the analyte into lowermolecular mass fragments before detection. Fragmentation can be achievedby any means known in the art. For example, protein analytes can befragmented using endoproteases. Carbohydrate analytes can be fragmentedusing glycosidases. Nucleic acids can be fragmented using endonucleases.The sample can be subject to fragmentation before or after docking withthe adsorbent.

J. Methods For Identifying Ligands For A Receptor

Functional pathways in biological systems frequently involve theinteraction between a receptor and a ligand. For example, the bindingbetween transcriptional activation frequently involves the prior bindingof a ligand with a transcription factor. Many pathological conditionsinvolve abnormal interaction between a receptor and its ligand.Interruption of the binding between a receptor and a ligand is afrequent target of drug discovery. However, the identity of a ligand fora receptor frequently is unknown, the receptor is an “orphan” receptor.

This invention provides a method using retentate chromatography toidentify ligands for receptors. The method involves docking a receptorto an adsorbent. Then, a sample that is suspected of containing a ligandfor the receptor is exposed to the docked receptor under an elutioncondition appropriate for binding between the receptor and the ligand.Then, ligands that have bound to the receptor are detected by desorptionspectrometry. The power of this method derives, in part, from thesensitivity to desorption spectrometry to detect small quantities ofmaterial docked to an adsorbent.

Docking the receptor to the adsorbent requires identifying an adsorbentthat retains, and preferably, specifically binds, the receptor. Methodsfor identifying adsorbents that specifically bind a protein aredescribed herein. In one method, the adsorbent comprises an antibodyspecific for the receptor. In another embodiment, the receptor isproduced as recombinant fusion protein that includes a moiety forspecific binding. For example, the receptor can be fused with the Fcportion of an antibody. Such portions bind to protein A which can beincorporated into an adsorbent.

The sample tested for the presence of a ligand is at the discretion ofthe practitioner. For example, if the receptor is a nuclear receptor,the sample can be nuclear extract. If the receptor is cytoplasmicreceptor, the sample can be cytoplasmic extract. If the receptor is acell surface receptor, the sample can be fluid from the surface to whichthe cell is exposed, for example, serum for an epithelial cell surfacereceptor.

The sample generally will be incubated with the receptor underphysiological conditions for a time sufficient to allow binding, forexample 37° C. for several hours. Then, unbound material is washed away.This method can quickly identify ligands that conventional techniquesrequire months to identify.

Retentate chromatography allows parallel processing of samples onseveral adsorbent sports. Accordingly, this method can involve testing aplurality of different samples for the presence of a ligand, as well asthe testing of a single sample under a plurality of incubation andelution conditions.

By determining the mass of the identified ligand and variousphysico-chemical properties, the ligand can be positively identifiedusing information from genome databases.

In another embodiment of this method a set of probes is prepared whichhas been exposed to and has docket proteins from a cell. This probe isuseful, itself, as a secondary probe to identify molecules from the cellthat bind to the docked molecules. After preparing a retentate map fromthe probe, the probe is secondarily exposed to the test material,generally under less stringent conditions than those used to prepare thesecondary probe, and the addressable locations analysed. Molecules thatare newly docked to the probe are those bound to the already-dockedmolecules.

K. Methods For Drug Discovery

Identifying molecules that intervene in the binding between a receptorand its ligand is an important step in developing drugs. This inventionprovides methods of screening compounds for their ability to modulatethe binding between an adsorbent and an analyte (e.g., a receptoradsorbent and a ligand analyte) by exposing an adsorbent and analyte toa test compound, and detecting binding between the adsorbent and theanalyte by description spectrometry.

Rapid screening of combinatorial libraries for drug candidates requiresthe ability to expose target interactions to thousands of drugs andidentify agents that interfere with or promote the interaction.Retentate chromatography enables one to dock one member of aligand/receptor pair to a substrate and to use it as a secondaryadsorbent. Then, after exposing the member up its partner and to theagent, one can determine by desorption spectrometry whether and to whatextent the partner has bound. Advantages of retentate chromatography inscreening methods include the ability to specifically dock the receptorto a substrate through an adsorbent, the ability to rapidly deploy thereceptor on many adsorbent spots for parallel processing, and the speedof throughput that is possible by reading results through desorptionspectrometry.

1. Screening Assay

The method involves providing an adsorbent; contacting the adsorbentwith the target analyte in the presence and absence of the agent underone or more selectivity conditions and determining whether the amount ofbinding with and without the agent. The amount of binding is determinedby retentate chromatography (e.g., by preparing a recognition profile).The experiment can be carried out with a control in which no agent isadded, or a control in which a different amount or type of agent isadded and the zero amount is determined by extrapolation. Astatistically significance difference in the amount of binding (p<0.05)indicates that the test agent modulates binding.

This method is particularly useful to screen analytes (e.g., proteins)as drug target candidates. After development of the protein retentionmap or recognition profile from serum or some other target cell type,the agent is exposed to the array of retained analyte at theiraddressable locations. After binding is allowed, unbound agent is elutedor washed away. Those analytes that retained bound agent under theselectivity conditions specified are identified directly by desorptionmass spectrometry, because the agent itself appears as a new componentin the retention map (i.e., the agent is desorbed and detecteddirectly). This method is particularly useful to screen drug candidates,both agonists and antagonists, for their ability to bind analytes ormodulate one or more biological processes.

2. Receptor and Ligand

The adsorbents and the target analyte need not engage in specificbinding. However, in particularly useful methods the adsorbent and thetarget analyte are a ligand/receptor pair.

In one embodiment, the ligand/receptor pair are a hormone and a cellsurface receptor or an intracellular receptor. The adsorbent can be anentire cell or cell membrane in the case of a membrane-bound receptor. Aprotein receptor or other drug target candidate may be used as anadsorbent to screen combinatorial drug libraries. Hundreds or thousandsof drug candidates can be applied to a single receptor type oraddressable location. After removal of unbound and weakly bound drugcandidates (i.e., agents) the bound agents are detected and identifiedby desorption spectrometry.

In another embodiment, the adsorbent is an enzyme that binds andmodifies the target substrate. The agents are screened for their abilityto modulate enzymatic transformation of the analyte. For example,enzymatic activity can be detected because the recognition profile of ananalyte may differ from that of the product of enzyme activity.Differential retention indicates that the agent alters binding.

The receptor/receptor can be retained on the substrate in a variety ofways. In one method, the receptor/ligand is directly retained by anon-specific adsorbent. In another method, the adsorbent is specific forthe receptor/ligand. For example, the adsorbent can contain an antibodyspecific for the receptor/ligand. The receptor/ligand can be a fusionprotein in which the fusion moiety binds the adsorbent, for example, inthe manner that an Fc fragment binds protein A. In one method, a geneticpackage, such as a phage from a phage display therapy, that has on itssurface a polypeptide that specifically binds the receptor/ligand, isbound to the substrate. The ligand is captured by the polypeptide. Also,the adsorbent can be an analyte already docked to the substrate, i.e.,it can be a secondary adsorbent, a tertiary adsorbent, etc.

This invention provides a plurality useful method to evaluate both thedirect and indirect consequences of drug (or other agent) binding to atarget. The detection of one or more analytes in a retentate mapgenerated from the proteins of a target cell type may be altered due tothe action of the agents (e.g., drug candidate) on 1) the target bindingprotein itself, 2) some other analyte (not the drug binding protein), or3) on gene expression (up or down regulation). It is the high resolvingand information generating power of retentate chromatography to detectthese changes, i.e., drug induced differences in the generic retentatemap or recognition profile observed with and without drug, that makesthis method one of the most powerful tools available tools available forproteomics, functional genomics, drug discovery, therapeutic drugmonitoring, and clinical diagnostics.

3. Test Agents

The choice of the agent to be tested is left to the discretion of thepractitioner. However, because of their variety and ease ofadministration as pharmaceuticals, small molecules are preferred as testagents.

a. Chemistry

The agent to be tested can be selected from a number of sources. Forexample, combinatorial libraries of molecules are available forscreening. Using such libraries, thousands of molecules can be screenedfor regulatory activity. In one preferred embodiment, high throughputscreening methods involve providing a library containing a large numberof potential therapeutic compounds (candidate compounds). Such“combinatorial chemical libraries” are then screened in one or moreassays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds” or can themselves be used as potential oractual therapeutics.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493, Houghton eta l. (1999) Nature, 354: 84-88). Peptide synthesisis by no means the only approach envisioned and intended for use withthe present invention. Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but notlimited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991),encode peptides (PCT Publication WO 93/20242, 14 Oct. 1993), randombio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261: 1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries, peptide nucleic acid libraries (see, e.g., U.S. Pat. No.5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996) NatureBiotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al. (1996) Science, 274: 1520-1522, andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum (1993) C&EN, Jan. 18, page 33, isoprenoidsU.S. Pat. No. 5,569,588, thiazolidinones and metathiazonones U.S. Pat.No. 5,549,974, pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134,morpholino compounds U.S. Pat. Nos. 5,506,337, benzodiazepines5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech. Louisville,Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass).

L. Methods For Generating Agents that Specifically Bind An Analyte

This invention provides methods for generating agents, e.g., singlechain antibodies, that specifically bind to a target analyte. Theseagents are useful, e.g., as specific diagnostic agents for dockingtargets in the study of ligand/receptor interactions. The method isparticularly useful for generating agents against targets that may onlybe isolated in such small quantities that it is not possible orpractical to generate antibodies by immunizing an animal. The methodinvolves the steps of providing a substrate having a target attachedthereto; providing a display library or genetic packages that displayagents to be screened; exposing the library to the target tospecifically retain genetic packages through interaction with the targetand detecting retained genetic packages by desorption spectrometry.

These steps can be conducted in parallel for a large number ofadsorbent-analyte candidates within complex populations without transferlosses and ambiguities associated with separate selection and detectionprocedures, including off-line amplification and labeling strategiesassociated with indirect detection means.

1. Providing the Substrate

The first step of the method involves providing a substrate thatcomprises an adsorbent that will serve as a target for a polypeptideagent of a display library to be screened. In one embodiment, thesubstrate is provided with the target adsorbent already attached. Inanother embodiment, the substrate is provided by providing a substratethat has an adsorbent that binds a target analyte, exposing theadsorbent to the analyte under elution conditions to allow retention ofthe analyte, and using the target adsorbent as the target for thedisplay library. In one embodiment, the target is differentiallyexpressed between two cell types that are being compared. For example,the targets may be derived from differentially expressed mRNA or may bedifferentially expressed polypeptides. Methods of identifying suchdifferentially expressed proteins by retentate chromatography methodsare described above.

Once a differentially expressed protein analyte is identified, one candevelop a selectivity condition that unambiguously resolves the analyte.More preferably, retention of the analyte is specific or exclusive. Themethods for progressive resolution of analytes described above make itpossible to identify selectivity conditions that specifically bind atarget analyte from a complex sample. In one embodiment, the boundtarget can be modified, e.g., by exposure to an enzyme.

Alternatively, the method can begin at the mRNA or EST stage. In thismethod, differentially expressed mRNAs or ESTs are identified by routinemethods. Then, these molecules are transcribed and translated in vitroand in situ on an adsorbent for docking. For example, a substrate fordesorption spectrometry having a plurality of adsorbent spots isprepared. The substrate is overlaid with a cylindrical tube, therebycreating a well with the adsorbent at the base of the well. In the wellone places reagents for in vitro transcription and translation of thedifferentially expressed mRNA (usually in the form of cDNA). (Formethods see, e.g., Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,(Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc.)) Translation of the mRNAor EST produces a polypeptide that is adsorbed. The cylindrical tube isremoved and the adsorbents spots are washed with an eluant, so as toidentify a selectivity condition that retains the polypeptide analyte.

2. Providing the Display Library

The second step involves providing a display library. The displaylibrary is comprised of genetic packages that display on their surfacesany sort of combinatorial library of peptides (“polypeptide agents”).However, single chain antibodies are attractive because they can be usedin subsequent immunoassays.

Many kinds of display libraries and their uses are known in the art. Abasic concept of display methods is the establishment of a physicalassociation between a polypeptide ligand to be screened and arecoverable polynucleotide that encodes the polypeptide. This physicalassociation is provided by a multimeric molecular complex, in this casethe genetic package, e.g., the phage particle, which displays apolypeptide as part of a capsid enclosing the phage genome which encodesthe polypeptide. The establishment of a physical association betweenpolypetides and their genetic material allows simultaneous massscreening of very large numbers of genetic packages bearing differentpolypeptides. Genetic packages displaying a polypeptide with affinity toa target bind to the target and these packages are enriched by affinityscreening to the target. The identity of polypeptides displayed fromthese packages can be determined from their respective genomes. Usingthese methods a polypeptide identified as having a binding affinity fora desired target can then be synthesized in bulk by conventional means.

The genetic packages most frequently used for display libraries arebacteriophage, particularly filementous phage, and especially phage M13,Fd and F1. Most work has inserted libraries encoding polypeptides to bedisplayed into either gIII or gVIII of these phage forming a fusionprotein. See, e.g., Dower, WO 91/19818; Devlin, WO 91/18989;MacCafferty, WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO92/18619 (gene VIII). See, also Cwirla et al., Proc. Natl. Acad. Sci.USA 87, 6378-6382 (1990); Devlin et al., Science 249, 404-406 (1990),Scott & Smith, Science 249, 386-388 (1990); Ladner et al., U.S. Pat. No.5,223,409 and Ladner et al., U.S. Pat. No. 5,571,698. Such a fusionprotein comprises a signal sequence, usually from a secreted proteinother than the phage coat protein, a polypeptide to be displayed andeither the gene III or gene VIII protein or a fragment thereof.Exogenous coding sequences are often inserted at or near the N-terminusof gene III or gene VIII although other insertion sites are possible.Some filamentous phage vectors have been engineered to produce a secondcopy of either gene III or gene VIII. In such vectors, exogenoussequences are inserted into only one of the two copies. Expression ofthe other copy effectively dilutes the proportion of fusion proteinincorporated into phage particles and can be advantageous in reducingselection against polypeptides deleterious to phage growth. Display ofantibody fragments on the surface of viruses which infect bacteria(bacteriophage or phage) makes it possible to produce human sFvs with awide range of affinities and kinetic characteristics.

In another variation, exogenous polypeptide sequences are cloned intophagemid vectors which encode a phage coat protein and phage packagingsequences but which are not capable of replication. Phagemids aretransfected into cells and packaged by infection with helper phage. Useof phagemid system also has the effect of diluting fusion proteinsformed from coat protein and displayed polypeptide with wild-type copiesof coat protein expressed from the helper phage. See, e.g., Garrard, WO92/09690.

Eukaryotic viruses can be used to display polypeptides in an analogousmanner. For example, display of human heregulin fused to gp70 of Moloneymurine leukemia virus has been reported by Han et al., Proc. Natl. Acad.Sci. USA 92, 9747-9751 (1995). Spores can also be used as replicablegenetic packages. In this case, polypeptides are displayed from theouter surface of the spore. For example, spores from B. subtilis havebeen reported to be suitable. Sequences of coat proteins of these sporesare provided by Donovan et al., J. Mol. Biol. 196, 1-10 (1987).

Cells can also be used as replicable genetic packages. Polypeptides tobe displayed are inserted into a gene encoding a cell protein that isexpressed on the cells surface. Bacterial cells including Salmonellatyphimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae,Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis,Bacterodes nodosus, Moraxella bovis, and especially Escherichia coli arepreferred. Details of outer-surface proteins are discussed by Ladner etal., U.S. Pat. No. 5,571,698, and Georgiou et al., Nature Biotechnology15, 29-34 (1997) and references cited therein. For example, the lamBprotein of E. coli is suitable.

3. Screening the Display Library

The third step involves screening the display library to identify aligand that specifically binds the target. The substrate bearing thetarget is exposed to the display library that displays polypeptideagents under elution conditions appropriate for specific binding betweenthe polypeptide and a target molecule. Genetic packages that have agentsthat recognize the target bind to the target already attached to thesubstrate. Removing unbound particles and retention of bound particlesresults from exposure to the elution condition.

A population of genetic packages, in this case M13 phage, representingapproximately 10¹¹ plaque forming units (pfu) per mL are introduced to asubstrate with an addressable array of bound target adsorbents (e.g.,protein). Upon contact, a selectivity condition optimized for the targetadsorbent (i.e., selectivity threshold modifier or eluent) is chosen,such that only a small subset of total phage genotypes are selectivelyretained, preferably fewer than 5-10. Note that unbound phage (i.e.,phage not bound to the target adsorbent) and phage loosely bound to thetarget adsorbent are eliminated by exposure to eluents that disrupt allbut the most selective analyte-target adsorbent interaction(s). Thosephage displaying polypeptides with the highest affinity for the targetadsorbent are selectively retained.

4. Detecting Bound Genetic Packages Containing Agents that SpecificallyBind the Target

In the fourth step, the binding of genetic packages to the target isdetected by desorption spectrometry. For example, the M13 phage hasthousands of copies of a single coat protein. Upon striking the phagewith a laser in desorption spectrometry, the coat proteins becomedislodged and are detectable. In this way, one can determine whether thelibrary contained a phage having an agent that bound to the target. Inorder to have genetic packages for subsequent analysis, the screeningstep can be performed in parallel at different locations on a probe, orthe substrate can have a physical dimension sufficiently large so thatthe laser does not dislodge all the genetic packages bound to thesurface. This method is particularly powerful, because even a few phagebound to the analyte can be detected.

In the case of M13, the preferred detection method is to monitor, bydesorption spectrometry, the appearance of the gene VIII coat protein asa “marker” protein signal. In this manner, we have detected “positive”target adsorbents with as few as 5 phage particles (pfu) bound (phageparticle number estimated by calculation from known dilutions). Otherphage markers, in order of preference, include gene V, gene X, and geneIII (including their fusion products).

After detection of those adsorbent locations with the highest affinityadsorbents, that is, those locations within the array with the fewestphage retained after exposure to high selectivity conditions (i.e.,stringent eluents), the bound package can now be used as a jump-offpoint for other uses.

5. Isolating the Genetic Package

In one embodiment, the method further involves isolating the geneticpackage for further analysis. This analysis can involve reproducing thegenetic package and isolating the polynucleotide from it. The isolatedphage are reproduced by the usual methods. For example, the retainedphage can be exposed to a biological amplification vehicle, for example,E. coli, plus nutritive media to grow the genetic packages forsubsequent analysis. Single clones can be further tested for ability tobind to analyte retained one substrate.

6. Sequencing the Nucleotide Sequence Encoding the Polypeptide Agent

Sequencing the nucleotide sequence encoding the polypeptide agent of abound genetic package provides information for producing the polypeptideagent. Sequencing can involve isolating the genetic package from theadsorbent, reproducing it, isolating the polynucleotide, and sequencingthe nucleotide sequence by any available means. In another method, thegenetic packages can be reproduced in situ by contacting the substratewith appropriate materials, such as cells subject to infection by thegenetic package. In another embodiment, sequencing is performed in situ.The method can involve lysing the genetic packages and amplifying thenucleotide sequence by any known means, e.g., PCR. Several differentgenetic packages may have bound to different epitopes available on thesurface. In this case, one may alter the elution conditions so that onlyone kind of phage binds to an epitope.

7. Producing the Polypeptide Agent

One valuable next step involves producing the polypeptide agent. Theisolated agent can be used, e.g., as an adsorbent for specific detectionof the target in diagnostics or for the study of ligand/receptorinteractions.

In one method, producing the polypeptide involves first sequencing thenucleotide sequence that encodes the polypeptide. The amino acidsequence can be derived from the nucleotide sequence. Sequencing can beaccomplished by the method as described above. The sequence can be thebasis for recombinant or chemical synthesis of the polypeptide agent.

In another method, the polypeptide can be produced by reproducing thegenetic package. This is particularly effective when the genetic packagecontains many copies of the polypeptide agent. The genetic package canbe reproduced in situ or after isolation.

A method of producing the polypeptide recombinantly can proceed asfollows. The nucleotide sequence encoding the polypeptide is eithersequenced or isolated by any means such as those discussed. Then, thenucleotide sequence is included in an expression vector. The expressionvector contains an expression control sequence operatively linked to thenucleotide sequence encoding the polypeptide. The expression vector canthen be used to express the polypeptide agent recombinantly by meanswell known in the art.

It is understood that the target can contain more than one epitope.Accordingly, the method can produce more than one polypeptide agentspecific for the target.

Target-specific agents can then be used as adsorbents for probes used inclinical diagnostics or drug discovery. That is, because such probescontain on their surface agents that specifically bind the target, theycan be used to isolate the target from complex mixtures, such asbiological samples, and to detect the target by desorption spectrometry.Furthermore, because the interaction between the agent and the targetcan be biospecific, it is likely to involve a greater affinity betweenthe two than an adsorbent developed by the progressive resolutionmethod, described above.

8. Isolating Peptide Epitopes of a Target

In one version this method allows one to isolate peptide epitopes of atarget analyte. The method employs an “anti-idiotypic”-like approach. Insummary, the epitopes of a target analyte are screened with, e.g., aphage display library. The isolated phage contain, e.g., single chainantibodies that recognize the epitopes of the analyte. These phage areused, in turn, to screen a second display library. The phage from thesecond library that bind to the single chain antibodies of the firstcontain displayed polypeptides that mimic the structure of the epitoperecognized by the single chain antibodies.

In one embodiment of this method, a nucleotide sequence encoding apolypeptide agent that binds the target analyte is used to produce M13phage in which the agent is displayed as a fusion with gene VIII. Thus,this phage has a coat with hundreds of copies of the target peptide onits surface. This phage is then docked to the adsorbent. Docking can beaccomplished through, e.g., a ligand that binds gene III, or gene IIIcan be modified to include a receptor for a ligand on the substrate. Thephage is then exposed to a second display library. Genetic packages fromthe library that bind to the docked phage are detected and isolated asdescribed. Preferably, the second display library contains a mass labelof some sort so that their gene VIII protein can be distinguished fromgene VIII of the phage docked to the substrate. Thus, the identificationof a substance as an “analyte target” or as an adsorbent can depend uponwhether the bound substance is used, subsequently, to bind anothersubstance. As one can see, the ability to bind a substance to an alreadydocked substance can continue, as can methods of identifying conditionsthat selectively remove the terminally bonded substance.

EXAMPLES

The following examples are offered by way of illustration, not by way oflimitation.

In the following examples, the following products and terms are employedChicken egg white lysozyme (1 μl diluted to 10 picomole/μl water), isavailable from Sigma Chemical Company, St. Louis, Mo. “Human serum”refers to a composition of 1 μl of human serum diluted 1 to 5 to 20 mMsodium phosphate buffer, 0.5 M NaCl, pH 7.0.

As used herein, “mg” means milligram(s): “ml” means milliliter(s); “μl”means microliter(s); “cm” means centimeter(s); “nm” means nanometer(s);“M” means molar; “mM” means millimolar; “min” means minute(s); “%” or“percent” is percent by weight unless otherwise specified; “NaCl” meanssodium chloride; “TFA” means trifluoroacetic acid.

1. PROTOCOLS FOR RETENTATE CHROMATOGRAPHY

The following protocols are examples of procedures for performingretentate chromatography.

A. Protocol for Retentate Mapping (using Chromatographic Series Array)

1. Sample Treatment

Dilute the biological sample (e.g., serum, urine, cell extract or cellculture medium) in 0.01% Triton X100 in HEPES or 20 mM Na phosphate, pH7.2. Centrifuge to clarify sample is necessary.

2. Sample Application

Add sample (1-5 μl) to a spot of Anionic, Normal phase or TED-Cu(II)adsorbent array. For a hydrophobic adsorbent array prewet each spot with0.5 μl acetonitrile containing 0.5% TFA. Add sample to the spot beforethe acetonitrile is dry.

Allow sample to concentrate (almost to dryness) on the spot.

3. Washing

a. Anionic Adsorbent Array

Wash spot 1 with 20 mM HEPES or Na phosphate. pH 7.2. Add the first 2 μlof wash solution to the spot before the sample is completely dry. Letthe wash solution sit on the spot for at least 15 sec. Pipet out and in10 times. Remove the first wash completely, repeat with the second washof 2 μl of the solution.

Wash spot 2 with 0.2 M NaCl in 20 mM Na phosphate, pH 7.2 as above.

Wash spot 3 with 1 M NaCl in 20 mM Na phosphate, pH 7.2 as above.

Wash spot 4 with 20 mM TrisHCl, pH 8.5 as above.

Wash spot 5 with 0.1 M Na acetate, pH 4.5 as above.

Wash spot 6 with 0.05% Triton X100 in 20 mM HEPES or Na phosphate, pH7.2 as above.

Wash spot 7 with 3 M urea in 20 mM HEPES or Na phosphate, pH 7.2 asabove.

Wash spot 8 with 10% acetonitrile in water as above.

Wash the whole array with water thoroughly.

Air dry the chip.

Add 0.3 μl Energy Absorbing Molecule (saturated solution prepared in 50%acetonitrile, 0.5% trifluoroacetic acid).

Air dry the chip.

Analyze the retained protein on each spot with laserdesorption/ionization time-of-flight mass spectrometer.

b. Normal Phase Adsorbent Array

Wash spot 1 with 5 mM HEPES, pH 7. Add the first 2 μl of wash solutionof the spot before the sample is completely dry. Let the wash solutionsit on the spot for at least 15 sec. Pipet out and in 10 times. Removethe first wash completely, repeat with the second wash of 2 μl of thesolution.

Wash spot 2 with 20 mM Na phosphate, 0.15 M NaCl, pH 7.2 as above.

Wash spot 3 with 20 mM Na phosphate, 0.5 M NaCl, pH 7.2 as above.

Wash spot 4 with 0.1 M Na acetate, pH 4.0 as above.

Wash spot 5 with 0.05% Triton X100 in 20 mM Na phosphate, 0.15 M NaCl,pH 7.2 as above.

Wash spot 6 with 3 M urea in 20 mM Na phosphate, 0.15 M NaCl, pH 7.2 asabove.

Wash spot 7 with 1% TFA as above.

Wash spot 8 with 30% isopropanol:acetonitrile (1:2) in water as above.

Wash the whole array with water thoroughly.

Air dry the chip.

Add 0.3 μEnergy Absorbing Molecule (saturated solution prepared in 50%acetonitrile, 0.5% trifluoroacetic acid).

Air dry the chip.

Analyze the retained protein on each spot with laserdesorption/ionization time-of-flight mass spectrometer.

c. TED-Cu(II) Adsorbent Array

Wash spot 1 with 20 mM Na phosphate, 0.5 M NaCl, pH 7.2. Add the first 2μl of wash solution to the spot before the sample is completely dry. Letthe wash solution sit on the spot for at least 15 sec. Pipet out and in10 times. Remove the first wash completely, repeat with the second washof 2 μl of the solution.

Wash spot 2 with 20 mM imidazole in 20 mM Na phosphate, 0.5 M NaCl, pH7.2 as above.

Wash spot 3 with 100 mM imidazole in 20 mM Na phosphate, 0.5 M NaCl, pH7.2 as above.

Wash spot 4 with 0.1 M Na acetate, 0.5 M NaCl, pH 4.0 as above.

Wash spot 5 with 0.05% Triton X100 in 20 mM Na phosphate, 0.15 M NaCl,pH 7.2 as above.

Wash spot 6 with 3 M urea in 20 mM Na phosphate, 0.15 M NaCl, pH 7.2 asabove.

Wash spot 7 with 1% TFA as above.

Wash spot 8 with 10% acetonitrile in water as above.

Wash the whole array with water thoroughly.

Air dry the chip.

Add 0.3 μl Energy Absorbing Molecule (saturated solution prepared in 50%acetonitrile, 0.5% trifluoroacetic acid).

Air dry the chip.

Analyze the retained protein on each spot with laserdesorption/ionization time-of-flight mass spectrometer.

d. Hydrophobic Adsorbent Array

Wash spot 1 with 5% acetonitrile in 0.1% TFA. Add the first 2 μl of washsolution to the spot before the sample is completely dry. Let the washsolution sit on the spot for at least 15 sec. Pipet out and in 10 times.Remove the first wash completely, repeat with the second wash of 2 μl ofthe solution.

Wash spot 2 with 50% acetonitrile in 0.1% TFA as above.

Wash spot 3 with 0.05% Triton X100 in 20 mM Na phosphate, 0.15 M NaCl,pH 7.2 as above.

Wash spot 4 with 3M urea in 20 mM Na phosphate, 0.15 M NaCl, pH 7.2 asabove.

Wash the whole array with water thoroughly.

Air dry the chip.

Add 0.3 μl Energy Absorbing Molecule (saturated solution prepared in 50%acetonitrile, 0.5% trifluoroacetic acid).

Air dry the chip.

Analyze the retained protein on each spot with laserdesorption/ionization time-of-flight mass spectrometer.

B. Protocol for Antibody-Antigen Assay; Receptor-Ligand Assay (usingPre-activated adsorbent array)

1. Immobilization of Antibody on Pre-activated Adsorbent Array

Place a pre-activated adsorbent array on a flat clean surface. Spot theantibody or receptor or control solution onto each spot of apre-activated adsorbent array prewetted with 0.5 μl of isopropanol (add1 μl antibody/spot before the isopropanol is dry).

Incubate (4° C. or room temperature, 2-18 h) in a humid chamber.

Use pipet to remove remaining solution from the spots.

Block residual active sites on the spots by adding 1 ml of 1 Methanolamine, pH 7.4 and PBS over the entire chip and incubate in ahumid chamber (room temperature 30 min.).

Wash the chip twice with 1% Triton X-100 in PBS. Submerge the chip inabout 9 ml of wash solution in a 15 ml conical plastic tube and rock onbenchtop agitator for at least 15 minutes.

Wash with 0.5 M NaCl in 0.1 M sodium acetate, pH 4.0 as above.

Wash with 0.5 M NaCl in 0.1 M TrisHCl, pH 8.0 as above.

Rinse with PBS as above. Then cover the chip with PBS and store at 4° C.until ready to use.

2. Binding of Antigen or Ligand

Gently shake or blot off PBS on the chip.

Add 1-5 μl of sample to each spot. For samples with very low antigen orligand concentration, put the adsorbent array into a bioprocessor. Washthe spots on the chip and Bioprocessor wells with 200 μl PBS two times.Add up to 300 μl of sample to each well.

Seal with adhesive tape.

Incubate with shaking (4° C. or room temperature, 1-18 h).

3. Washing

Remove sample from the spots, wash each spot with 2 μl of 0.1% TritonX100 in PBS, pH 7.2, two times. Add the first 2 μl of wash solution tothe spot. Let the wash solution sit on the spot for at least 15 sec.Pipet out and in 10 times. Remove the first wash completely, repeat withthe second wash of 2 μl of the solution. This is followed by a wash with0.5 M NaCl in 0.1 M HEPES, pH 7.4.

Wash the whole array with water thoroughly.

4. Analysis of Retained Proteins

Air dry the chip.

Add 0.3 μl Energy Absorbing Molecule (saturated solution of SinapinicAcid or EAMI or CHCA prepared in 50% acetonitrile, 0.5% trifluoroaceticacid).

Air dry the chip.

Analyze the retained protein on each spot with laserdesorption/ionization time-of-flight mass spectrometer.

II. RECOGNITION PROFILE OF LYSOZYME

We generated a recognition profile for lysozyme using high-informationresolution retentate chromatography. The profile includes resolution oflysozyme with six adsorbents, each under a variety of differentselectivity threshold modifiers. The result is 40 differentspectrographs that differently characterize the physico-chemicalproperties of lysozyme.

A. Lysozyme Recognition Profile Using a Hydrophilic Adsorbent Array

Chicken egg white lysozyme is added to various spots of achromatographic series adsorbent array of a silicon oxide adsorbent on astainless steel substrate. After incubation in a moist chamber at roomtemperature for 15 min., each different spot of adsorbent is washed withone of the following eluants (selectivity threshold modifiers):

(1) 20 mM sodium phosphate buffer, pH 7.0,

(2) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(3) 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(4) 25 mM sodium acetate buffer, 0.125 M NaCl, pH 4.5,

(5) 1% TFA,

(6) 10% acetonitrile in water,

(7) 20% acetonitrile in water,

(8) 0.05% Tween20 in 20 mM sodium buffer, 0.15 M NaCl, pH 7.0, and

(9) 3M urea in 20 mM sodium phosphate buffer, pH 7.0.

Each wash includes pipetting 1 μl of wash solution in and out of thespot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer, using a nitrogen laser(355 nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c (available from Galactic Industries Corporation)for data overlay presentation.

FIG. 5A shows the composite mass spectrum of a lysozyme recognitionprofile on a normal phase chromatographic series adsorbent array. Thebottom profile shows the lysozyme signal intensity retained on thesilicon oxide adsorbent after washing with pH 7 buffer alone. Inclusionof sodium chloride (0.2-0.4 M) in the selectivity threshold modifierdecreases the retention of lysozyme. This indicates that the interactionof lysozyme (a basic protein) with a silicon oxide (negatively chargedat pH 7) adsorbent involves an ion exchange mechanism. Lowering the pHof the selectivity threshold modifier, for example to pH 4.5 in thesodium acetate buffer, or <2 in 1% TFA, almost completely eliminates thenegative charge on the silicon oxide adsorbent, and lysozyme is notretained any longer. Including polarity modulating agents, (e.g.,organic solvents (e.g., acetonitrile), or detergent (e.g., Tween20), orurea in the selectivity threshold modifier also reduces the interactionof lysozyme with the silicon oxide adsorbent. This indicates that theother interaction mechanism involves a hydrophilic interaction.

B. Lysozyme Recognition Profile Using a Hydrophobic Adsorbent Array

Chicken egg white lysozyme is added to various spots of achromatographic series adsorbent array of polypropylene (C₃ hydrophobic)adsorbent coated on silicon oxide-coated stainless steel substrate.After incubation in a moist chamber at room temperature for 15 min.,each different spot of adsorbent is washed with one of the followingeluants (selectivity threshold modifiers):

(1) 0.1% TFA,

(2) 10% acetonitrile in 0.1% TFA,

(3) 20% acetonitrile in 0.1% TFA,

(4) 50% acetonitrile in 0.1% TFA,

(5) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0,and

(6) 3M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0.

Each wash includes pipetting 1 μl of wash solution in and out of thespot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Afterwards, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TGA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

FIG. 5B shows the composite mass spectrum of lysozyme recognitionprofile on a hydrophobic C₃ chromatographic series adsorbent array. Thebottom profile shows the lysozyme signal intensity retained on thehydrophibic C₃ adsorbent after washing with 0.1% TFA alone. Including apolarity of modulating agent, (e.g., acetonitrile) in the selectivelythreshold modifier decreases the retention of lysozyme on thehydrophobic C₃ adsorbent. The acetonitrile concentration range forelution of lysozyme from the hydrophobic C₃ absorbent is between 20-50%.Including detergent (Tween20), or urea, in the selectivity thresholdmodifier does not significantly reduce the retention of lysozyme on thehydrophobic C₃ adsorbent.

C. Lysozyme Recognition Profile Using a Phenyl Hydrophobic AbsorbentArray

Chicken egg white lysozyme is added to various spots of an adsorbentarray of polystyrene (phenyl hydrophobic) adsorbent coated on siliconoxide-coated stainless steel substrate. After incubation in a moistchamber at room temperature for 15 min., one spot of adsorbent is washedwith one of the following eluants (selectivity threshold modifiers):

(1) 0.1% TFA,

(2) 10% acetonitrile in 0.1% TFA,

(3) 20% acetonitrile in 0.1% TFA,

(4) 50% acetonitrile in 0.1% TFA,

(5) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0,and

(6) 3M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0.

Each wash includes pipetting 1 μl of wash solution in and out of thespot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355mm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

FIG. 5C shows the composite mass spectrum of the lysozyme recognitionprofile on the hydrophobic phenyl chromatographic series adsorbentarray. The bottom profile shows the lysozyme signal intensity retainedon the hydrophobic phenyl absorbent after washing with 0.1% TFA alone.Including a polarity modulating agent, (e.g., acetonitrile) in theselectivity threshold modifier decreases the retention of lysozyme. Theacetonitrile concentration range for elution of lysozyme from thehydrophobic C₃ adsorbent is between 20-50%, however, when the lysozymepeak intensities retained on the C₃ and phenyl surface are comparedunder the same 20% acetonitrile wash condition, the interaction oflysozyme with the phenyl adsorbent is less strong. Including detergent(e.g., Tween20), or urea, in the selectivity threshold modifier alsosignificant reduces the retention of lysozyme on the hydrophobic phenyladsorbent.

D. Lysozyme Recognition Profile Using an Anionic Adsorbent Array

Chicken egg white lysozyme is added to various spots of an adsorbentarray of anionic group (SO₃ ⁻) adsorbent (i.e., a cationic exchangeadsorbent) coated on silicon oxide-coated stainless steel substrate.After incubation in a moist chamber at room temperature for 15 min.,each different spot of adsorbent is washed with one of the followingeluants (selectivity threshold modifiers):

(1) 20 mM sodium phosphate buffer, pH 7.0,

(2) 0.1 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(3) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(4) 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(5) 25 mM sodium acetate buffer, 0.125 M NaCl, pH 4.5,

(6) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0,and

(7) 3M urea in 20 mM sodium phosphate buffer, pH 7.0.

Each wash includes pipetting 1 μl of wash solution in and out of thespot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355mm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

FIG. 5D shows the composite mass spectrum of the lysozyme recognitionprofile on a cation exchange chromatographic series array. The bottomprofile shows the lysozyme signal intensity retained on the anionicadsorbent after washing with pH 7 buffer alone. Including increasingconcentrations of sodium chloride (0.1-0.4 M) in the selectivitythreshold modifier decreases the retention of lysozyme. This indicatesthat the interaction of lysozyme (a basic protein) with the anionicadsorbent involves an ion exchange mechanism. A 0.4 M NaCl concentrationis required to elute the lysozyme. Lowering the pH of the selectivitythreshold modifier to pH 4.5 in the sodium acetate buffer, does notaffect the retention of lysozyme on a strong anionic adsorbent.Including a polarity modulating agent (e.g., a detergent such asTween20, or urea) in the selectivity threshold modifier reduces theinteraction of lysozyme with an anionic adsorbent. This indicates thatthe interaction of a hydrophobic lysozyme protein with the anionicadsorbent is modulated by the polarity of the eluant.

E. Lysozyme Recognition Profile Using an Cationic Absorbent Array

Chicken egg white lysozyme is added to various spots of an adsorbentarray of cationic (quaternary amine) adsorbent coated on siliconoxide-coated stainless steel substrate. After incubation in a moistchamber at room temperature for 15 min., each different spot ofadsorbent is washed with one of the following eluants (selectivitythreshold modifiers):

(1) 20 mM sodium phosphate buffer, pH 7.0,

(2) 0.1 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(3) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(4) 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(5) 25 mM sodium acetate buffer, 0.125 M NaCl, pH 4.5,

(6) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0,or

(7) 3M urea in 20 mM sodium phosphate buffer, pH 7.0.

Each wash includes pipetting 1 μl of wash solution in and out of thespot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

FIG. 5E shows the composite mass spectrum of the lysozyme recognitionprofile on the cationic (anion exchange) adsorbent chromatographicseries adsorbent array. The retention of the basic lysozyme protein onthe cationic adsorbent is very weak. The effect of modulating theselectivity threshold modifiers on lysozyme retention is minimal.

F. Lysozyme Recognition Profile Using an Immobilized Metal Ion AdsorbentArray

Chicken egg white lysozyme is added to various spots of an adsorbentarray of immobilized metal (iminodiacetate-Cu) adsorbent coated onsilicon oxide-coated stainless steel substrate. After incubation in amoist chamber at room temperature for 15 min., each different spot ofadsorbent is washed with one of the following eluants (selectivitythreshold modifiers):

(1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,

(2) 5 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,

(3) 0.1 M sodium acetate buffer, 0.5 M NaCl, pH 4.5.

(4) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0,or

(5) 3M urea in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

Each wash includes pipetting 1 μl of wash solution in and out of thespot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

FIG. 5F shows the composite mass spectrum of the lysozyme recognitionprofile on the immobilized metal chromatographic series adsorbent array.The bottom profile shows the lysozyme signal intensity retained on theimmobilized copper ion adsorbent after washing with pH 7 buffer alone.Including a histidine-binding competitive affinity ligand (e.g.,imidazole) in the selectivity threshold modifier eliminates theretention of lysozyme. This indicates that the interaction of lysozyme(which has a single histidine residue in the sequence) with animmobilized copper ion adsorbent involves a coordinate covalent bindingmechanism. Lowering the pH of the selectivity threshold modifier to pH4.5 in the sodium acetate buffer, also decreases the retention oflysozyme on the immobilized copper adsorbent. It is believed that thisis a result of the protonation of the histidine residue on lysozyme,which inhibits the coordinate covalent interaction. Including detergent(i.e. Tween20) does not affect the interaction. Including ureacompletely eliminates the retention of lysozyme on the immobilizedcopper adsorbent.

III. RESOLUTION OF ANALYTES IN HUMAN SERUM

We resolved analytes in human serum using a variety of adsorbents andeluants. These results show that analytes are differentially retained bydifferent adsorbents, and that retention chromatography is able toprovide information at both low and high molecular masses.

A. Human Serum Protein Recognition Profile Using an Immobilized MetalIon Adsorbent Array

Human serum is added to various spots of an adsorbent array ofimmobilized metal ion (tri(carboxymethyl)ethylenediamine-Cu) adsorbentcoated on silicon oxide-coated stainless steel substrate. Afterincubation in a moist chamber at room temperature for 15 min., eachdifferent spot of adsorbent is washed with one of the following eluants(selectivity threshold modifiers):

(1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,

(2) 5 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,

(3) 0.1 M sodium acetate buffer, 0.5 M NaCl, pH 4.5,

(4) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0,and

(5) 3M urea in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

Each wash includes pipetting 1 μl of wash solution in and out of thespot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

FIGS. 6A and 6B shows the composite mass spectrum at low and highmolecular mass of the serum protein recognition profile on theimmobilized metal chromatographic series adsorbent array. The bottomprofile shows the serum proteins retained on the immobilized copperadsorbent after washing with pH 7 buffer alone. Including ahistidine-binding competitive affinity ligand (e.g., imidazole), ordetergent (e.g., Tween20), or urea in the selectivity thresholdmodifier, or lowering the pH of the selectivity threshold modifier to4.5, differentially enhances or decreases the retention of differentcomponents of the complex protein mixture on the same adsorbent.

B. Human Serum Protein Recognition Profile Using a Plurality ofDifferent Adsorbents

Human serum is added to various spots of an adsorbent array of thefollowing different adsorbents:

(1) C₃ hydrophobic;

(2) phenyl hydrophobic,

(3) anion exchange,

(4) cation exchange, and

(5) immobilized metal (tri(carboxymethyl)ethylenediamine-Cu).

Each adsorbent is coated on a silicon oxide-coated stainless steelsubstrate. After incubation in a moist chamber at room temperature for15 min., each spot of adsorbent is washed with 0.05% Tween20 in 20 mMsodium phosphate buffer, 0.15 M NaCl, pH 7.0 as the selectivitythreshold modifier.

Each wash includes pipetting 1 μl of wash solution in and out of thespot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile 0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

FIG. 7A and 7B show the composite mass spectrum of the serum proteinrecognition profile on various adsorbents of a chromatographic seriesadsorbent array. The use of a single selectivity threshold modifier on aplurality of different adsorbents (having different bindingcharacteristics) differentially enhances or decreases the retention ofdifferent components of the complex protein mixture on the differentadsorbents.

IV. RESOLUTION OF ANALYTES IN PRETERM INFANT URINE

We resolved analytes in preterm infant urine using a variety ofadsorbents and eluants. These results show that because adsorbentsretain analytes differentially, the use of various adsorbents providesgreat resolving ability. They also show the ability to identifyadsorbents that preferentially retain specific analytes, which is usefulfor developing purification schemes.

A. Resolution of Analytes in Preterm Infant Urine Using A Variety ofAdsorbents and the Same Eluant (Water)

Preterm infant urine (2 μl) is added to various spots of a carbonizedPEEK polymer substrate coated with the following different adsorbents:

(1) C₈ hydrophobic (Octyl Sepharose, available from Sigma),

(2) phenyl hydrophobic (Phenyl Sepharose, available from Sigma),

(3) anion exchange (Q Sepharose, available from Sigma),

(4) cation exchange (S Sepharose, available from Sigma),

(5) immobilized metal (IDA-Cu, Chelating Sepharose, available fromPharmacia), and

(6) immobilized metal (tri(carboxymethyl)ethylenediamine-Cu Sepharose).

After incubation in a moist chamber at room temperature for 15 min.,each spot of adsorbent is washed with water as the selectivity thresholdmodifier. Each wash includes pipetting 1 μl of wash solution in and outof the adsorbent three times. This is repeated with a fresh aliquot ofwash solution. An aliquot of 0.3 μl of sinapinic acid (5 mg/ml 50%acetonitrile:0.5% TFA) is added and allowed to air dry. The array isanalyzed with the a laser desorption/ionization time-of-flight massspectrometer from Hewlett Packard (Model 2030) that uses a nitrogenlaser (355 mm) and a 150 cm flight tube. The data is analyzed by HPMALDI TOF software and exported to GRAMS/32c for data overlaypresentation.

FIGS. 8A and 8B show the composite mass spectrum at low and highmolecular mass of the preterm infant urine protein recognition profileon the various adsorbents of a chromatographic series. The use of asingle selectivity threshold modifier (i.e., water) on the variousadsorbents (each having a different binding characteristic)differentially enhances or decreases the retention of differentcomponents of the complex protein mixture like on the differentadsorbents.

B. Resolution of Analytes in Preterm Infant Urine Using a HydrophobicPhenyl Adsorbent Indirectly Coupled to the Substrate and Three DifferentEluants

Preterm infant urine (2 μl) is added to various spots of a carbonizedPEEK polymer substrate coated with phenyl hydrophobic adsorbent (PhenylSepharose, available from Sigma). After incubation in a moist chamber atroom temperature for 15 mins., each spot of adsorbent is washed with oneof the following eluants (selectivity threshold modifiers):

(1) water,

(2) 2M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, and

(3) 0.1% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0.

Each wash includes pipetting 1 μl of wash solution in and out of thespot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with a laser desorption/ionization time-of-flight massspectrometer from Hewlett Packard (model 2030) that uses a nitrogenlaser (355 nm) and a 150 cm flight tube. The data is analyzed by HPMALDI TOF software and exported to GRAMS/32c for data overlaypresentation.

FIG. 9 shows the composite mass spectrum of the preterm infant urineprotein recognition profile on the hydrophobic phenyl adsorbent of achromatographic series. The application of various eluants havingdifferent elution characteristics on a single adsorbent differentiallyenhances or decreases the retention of different components of thecomplex protein mixture. One of the components (marked by *) isselectively retained on the hydrophobic phenyl adsorbent when 01%Tween20 in PBS is used as the eluant.

V. IDENTIFICATION OF PROTEINS IN CULTURE MEDIUM FROM TWO DIFFERENT CELLLIVES

This example illustrates the identification of proteins that aredifferentially expressed in cells with adsorbent array: Chromatographicseries.

Two different breast cancer cell lines are cultured for the same periodof time in a constant composition culture medium. After concentrationwith a filtration unit, an aliquot of 1 μl of each culture medium isadded to various spots of a an adsorbent array (Ciphergen Biosystems,Inc., Palo Alto, Calif.) of immobilized metal(tri(carboxymethyl)ethylenediamine-Cu) adsorbent coated on siliconoxide-coated stainless steel as substrate. After incubation at roomtemperature in a moist chamber of 15 min., a spot of adsorbent is washedwith either one of the following eluants (selectivity thresholdmodifiers).

(1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,

(2) 20 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH7.0,

(3) 0.1 M sodium acetate buffer, 0.5 M NaCl, pH 4.5,

(4) 0.1% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0,

(5) 3M urea in 10 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0, or

(6) 1% TFA.

Each wash includes pipetting 1 μl of wash solution in and out of thespot of adsorbent three times. This is repeated with a fresh aliquot ofwash solution. Afterwards, the spot of adsorbent is washed with 1 μl ofwater two times. An aliquot of 0.3 μl of sinapinic acid (5 mg/ml 50%acetonitrile:0.5% trifluoroacetic acid) is added and allowed to air dry.The array is analyzed with a laser desorption/ionization time-of-flightmass spectrometer that uses a nitrogen laser (355 nm) and a 60 cm flighttube. The data is analyzed by computer and exported to GRAMS/32c(Galactic Industries Corporation) for data overlay presentation.

FIG. 10A shows the composite mass spectrum of cell secreted proteinrecognition profile of cell line 1 on an immobilized metal (Cu)chromatographic series adsorbent array. The application of variouseluants of different selectivity thresholds on a single adsorbentdifferentially enhances or decreases the retention of differentcomponents of a complex protein mixture like cell culture medium.

FIG. 10B shows the composite mass spectrum of cell secreted proteinrecognition profiles of both cell lines on an immobilized metal (Cu)chromatographic series adsorbent array. The same eluant, 0.1% Tween20+3M urea in 10 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0, is used towash away unretained materials. The peak marked 7532 Da is the majorretained peak in cell line 1 secreted protein is not expressed in cellline 2.

FIG. 10C shows the composite mass spectrum of cell secreted proteinrecognition profiles of cell line 1 on an immobilized metal (Ni)chromatographic series adsorbent array. Using the same eluant, 0.1%Tween20+3 M urea in 10 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,but employing an adsorbent of different surface interaction potential(i.e., immobilized Ni metal vs immobilized Cu metal), the 7532 Da peakis the only retained protein among all the cell line 1 secretedproteins. The inset shows the same mass spectrum on an expanded scale.The smaller peak at 3766 Da is the doubly charged species of the sameprotein.

FIG. 10D shows the composite mass spectrum of cell secreted proteinrecognition profiles of cell line 1 on an immobilized metal (Ni)chromatographic series adsorbent array before (lower profile) and after(top profile) in situ trypsin digestion. The peptide map generated for apure protein is a fingerprint of that protein and can be used foridentification.

VI. COMPARISON OF RETENTATE CHROMATOGRAPHY WITH 2D GEL ELECTROPHORESIS

One advantage of retentate chromatography is the ability to rapidlyresolve analytes in a variety of dimensions, resulting in highinformation content about a variety of physico-chemical characteristics.In contrast, 2D gel electrophoresis provides resolution in twodimensions only.

FIG. 11 shows a preterm infant urine protein recognition profile onphenyl hydrophobic adsorbent of a chromatographic series. Theapplication of various eluants and adsorbents yields multi-dimensionalinformation. The use of different selectivity conditions differentiallyenhances or decreases the retention of various components of a complexprotein mixture (such as preterm infant urine), resulting in detailedresolution of analytes.

In contrast, FIG. 12 shows a two-dimensional separation of proteins inpreterm infant urine according to pI and molecular mass. The gelprovides information about two dimensions, only, as compared to the sixdimensions used as adsorbents in retentate chromatography. Spots are notas well resolved as by mass spectrometry and resolution at very high andvery low molecular masses is limited.

VII. SEQUENTIAL EXTRACTION OF ANALYTES FROM A SAMPLE

Analytes can be sequentially extracted from a sample by seriallyexposing the sample to a selectivity condition followed by collection ofthe un-retained sample.

A Hemophilus knockout mutant lysate was prepared in 10% glycerol 50 mMEDTA. After centrifugation, the supernate was diluted 1:3 in 0.01%Triton X100 in 25 mM HEPES, pH 7.4. An aliquot of 2 μl of the dilutedsample was added to a spot of an adsorbent array anionic site. Afterinculation at room temperature for 30 min, the remaining sample on theanionic site was transferred to a spot of adsorbent array normal phasesite. The spot of anionic site was washed with 2 μl of 0.01% Triton X10025 mM HEPES two times. Each wash was accomplished by pipetting the washsolution in and out of the spot ten times. The washes were combined withthe sample initially added on the normal phase spot.

After incubation at room temperature for 30 min, the remaining sample onthe normal phase site was transferred to a spot of adsorbent arrayNi(II) site. The spot of normal phase site was washed with 2 μl ofphosphate buffered saline two times. Each wash was accomplished bypipetting the wash solution in and out of the spot ten times. The washeswere combined with the sample initially added on the Ni(II) spot.

After the sample was concentrated to near dryness on the Ni(II) spot,the unbound analytes were recovered by washing with 2 μl of 100 mMimidazole in phosphate buffered saline two times. Each wash wasaccomplished by pipetting the wash solution in and out of the spot tentimes. The washes were transferred to a spot of adsorbent arrayaliphatic hydrophobic site.

The sample was allowed to concentrated to near dryness on thehydrophobic site, unbound analytes were removed by washing with 2 μl of5% acetonitrile in 0.1% trifluoroacetic acid two times. Each wash wasaccomplished by pipetting the wash solution in and out of the spot tentimes.

Each spot of anionic, normal phase, Ni(II), and hydrophobic site waswashed with 2 μl of water to remove remaining buffer. An aliquot of 0.3μl of sinapinic acid solution in 50% acetonitrile 0.5% trifluoroaceticacid was added to each spot. The retained analytes on each site wasanalyzed with laser desorption/ionization time-of-flight massspectrometer.

FIGS. 19A-19D show the retention map of Hemophilus lysate on adsorbentarray. Multiple peaks in the mass range 3000 to 25000 Da were observedon the adsorbents. Note that each adsorbent shows different retentionfor each of the analytes in the sample.

VIII. PROGRESSIVE RESOLUTION OF AN ANALYTE

By adding new binding or elution characteristics to a selectivelycondition that resolves an analyte, one can develop a selectivitycondition that provides improved resolution of the analyte. In thisexample, a sample was bound to a Cu(II) adsorbent and exposed to a firsteluant and two second eluants. The second eluants differed from thefirst by the addition of another elution condition. Each added conditionimproved resolution of the analyte.

Hemophilus wild type stationary phase lysate prepared in 10% glycerolwas diluted 1:1 in 20 mM sodium phosphate, 0.5 M sodium chloride, pH7.0. After centrifugation, an aliquot of 150 μl of the supernate wasincubated with each spot of adsorbent array Cu(II) site in abioprocessor. After mixing in the cold for 30 min, the sample wasremoved. Each spot was washed with a different lysate. A first spot waswashed with 150 μl of 20 mM sodium phosphate, 0.5 M sodium chloride, pH7.0. A second spot was washed with 150 μl of 0.05% Triton X100 inaddition to 20 mM sodium phosphate, 0.15 M NaCl, pH 7.0. A third spotwas washed with 150 μl of 100 mM imidazole in addition to 20 mM sodiumphosphate, 0.15 M NaCl, pH 7.0. Each wash was accomplished by incubatingthe wash solution with the spot for 5 min with mixing. The wash wasrepeated two times. Each spot was washed with water to remove detergentand buffer.

The adsorbent array was removed from the bioprocessor. An aliquot of 0.3μl of sinapinic acid solution in 50% acetonitrile 0.5% trifluoroaceticacid was added to each spot. The retained analytes on each spot wasanalyzed with laser desorption/ionization time-of-flight massspectrometer.

FIGS. 20A-20C show the retention map of Hemophilus lysate on adsorbentarray Cu(II) site after washing under the three elution conditionsdescribed above. Multiple peaks in the mass range 2000 to 18000 Da wereobserved. The protein marked with a “*” was only a minor component inthe retention map of FIG. 20A. When the selectivity condition wasmodified by the addition of a detergent, Triton X100 (FIG. 20B), to thesame buffer, the same protein “*” was retained better than the otheranalytes and resolved better. When the selectivity condition wasmodified by the addition of an affinity displacer, imidazole, to thesame buffer (FIG. 20C), the protein “*” was highly resolved from theother analytes in the retentate map.

This strategy of progressive identification of selectivity conditionswith improved resolution for an analyte can be adopted to develop amethod for the preparative purification of this protein from the totalHemophilus lysate.

IX. DIFFERENTIAL EXPRESSION OF AN ANALYTE: MARKER PROTEIN DISCOVERY

A. Human serum

An aliquot of 0.5 μl of normal or diseased human sera was diluted withan equal volume of 20 mM sodium phosphate, 0.5 M NaCl, pH 7.0. Each wasapplied to a different spot on an adsorbent array Cu(II) site. Afterincubation at 4° C. for 1 h, each spot was washed with 2 μl of 20 mMsodium phosphate, 0.5 M NaCl, pH 7.0, two times. Each wash wasaccomplished by pipetting the wash solution in and out of the spot tentimes. Each spot was finally washed with 2 μl of water to removeremaining buffer. An aliquot of 0.3 μl of sinapinic acid solution in 50%acetonitrile 0.5% trifluoroacetic acid was added to each spot. Theretained analytes on each spot was analyzed with laserdesorption/ionization time-of-flight mass spectrometer.

Proteins marked with a “*” in FIG. 21D are present in significantlygreater amounts in diseased serum than in normal serum. The resultsillustrate a method for discovery of disease markers that can be used inclinical diagnostics.

B. Mouse urine

An aliquot of 1 μl of normal, diseased or drug treated mouse urine wasapplied to a different spot of an adsorbent array Cu(II) site. Afterincubation at room temperature for 10 min, each spot was washed with 2μl of 100 mM imidazole in 20 mM sodium phosphate. 0.15 M NaCl, pH 7.0,two times. Each wash was accomplished by pipeting the wash solution inand out of the spot ten times. Each spot was finally washed with 2 μl ofwater to remove remaining buffer. An aliquot of 0.3 μl of sinapinic acidsolution in 50% acetonitrile 0.5% trifluoroacetic acid was added to eachspot. The retained analytes on each spot was analyzed with laserdescription/ionization time-of-flight mass spectrometer.

The retentate maps of normal (control), diseased and drug treated mouseurine are shown in the FIG. 1. One analyte was found to be present inmuch higher quantity in the disease mouse urine (middle panel), the sameanalyte was not found in normal mouse urine (upper panel), and found indrug treated mouse urine in much reduced quantity (lower panel). Thisanalyte can be used as a potential disease marker. To illustrate thefeasibility of a quantitative diagnostic assay, the area under the peakof the retained marker protein are calculated and shown in the table. Aclear quantitative difference is observed between the disease and drugtreated mouse urines. To compensate for experimental variability, aninternal standard analyte was used. The normalized disease marker peakarea (i.e., peak area of marker divided by peak area of internalstandard) for each urine sample is presented in the bottom panel. Thereis at least a ten fold reduction of the disease urine marker after drugtreatment.

C. Human Urine

Urines from normal human and cancer patients were diluted 1:2 in 0.01%TRITON X100™(t-octylphenoxypolyehoxyethanol) in phosphate bufferedsaline. An aliquot of 1.5 μl of normal or disease human urine wasapplied to a different spot of an adsorbent array aliphatic hydrophobicsite prewetted with 0.5 μl of isopropanol/acetonitrile (1.2) 0.1%trifluoroacetic acid. After incubation at 4 C for 30 min, each spot waswashed with 2 μl of 50% ethylene glycol in 10 mM TrisHCl, 0.05 M NaCl,pH 7.5, two times. Each wash was accomplished by pipeting the washsolution in and out of the spot ten times. Each spot was finally washedwith 2 μl of water to remove remaining ethylene glycol and buffer. Analiquot of 0.3 μl of sinapinic acid solution in 50% acetonitrile 0.5%trifluoroacetic acid was added to each spot. The retained analytes oneach spot was analyzed with laser desorption/ionization time-of-flightmass spectrometer.

The retentate maps of urines of four cancer patients and a normal humanare shown FIG. 23A. Multiple protein peaks were retained on theadsorbent array hydrophobic site after washing with 50% ethylene glycolin Tris/NaCl buffer. To identify possible disease markers, differencemaps between individual patient urine and normal urine are plotted. Eachbar in the difference plot above the baseline represents an analytepresent in higher quantity in the patient urine. (FIG. 23B-23D.)Variations in the patterns of difference map of the patients reflectindividual fluctuations in a population. However, one analyte around5000 Da (marked with *) and a cluster of analytes around 7500 Da (markedwith *), are found to be consistently present in higher quantities inall patients, therefore these can be identified as potential diseasemarkers.

X. CAPTURE OF PHAGE FROM PHAGE DISPLAY LIBRARY

Viruses adsorbed to the surface of a protein chip can be detected bydesorption spectrometry. Antibodies against viral coat proteins, used asadsorbents, can capture viruses. A target protein used as an absorbentcan capture phage displaying a single-chain antibody against the target.

A. Detection sensitivity of Phage Display Antibody by AdsorbentSubstrate

M13 phage (10¹² particle/ml) in growth medium was serially diluted into0.01% TRITON X100™ in 25 mM HEPES, pH 7.4. An aliquot of 0.25 μl of eachof the diluted phage suspension was added to a spot of an adsorbentarray aliphatic hydrophobic site. An aliquot of 0.3 μl of Cycad hydroxycinnamic acid (“CHCA”) in 50% acetonitrile, 0.5% trifluoroacetic acidwas added. The samples were analyzed by laser desorption/ionizationtime-of-flight mass spectrometer.

The M13 phage Gene VIII protein was detected with high sensitivity onthe array. FIGS. 24A-24E. A detectable signal (signal/noise>2) wasobtained when the phage suspension was diluted 10,000,000 times.

B. Identification of M13 Phage by Adsorbent Array

Rabbit anti-M13 antibody (Strategene) was immobilized on Protein A HyperD (BioSepra), and washed with phosphate buffered saline, pH 7extensively. An aliquot of 1-10 μl suspension of M13 phage (10¹²particle/ml) in growth medium was incubated with 1 μl aliquot ofimmobilized anti-M13 antibody at 4° C. overnight. After washing with0.05% Tween 20 in phosphate buffered saline, pH 7 and then with water toremove detergent and buffer, an aliquot of the captured phage wasanalyzed with laser desorption/ionization time-of-flight massspectrometer in the presence of sinapinic acid.

The anti-M13 antibody control shows only the antibody signal (singly anddoubly charged). FIG. 25A. When the M13 phage was captured by theantibody, the most easily identifiable protein peaks from the phage arethe Gene VIII protein and the Gene III protein fusion with single chainantibody. FIG. 25B. Since the M13 phage Gene VIII protein is detectedwith high efficiency by the method, it can be used as a sensitivemonitor of phage capture.

C. Specific Capture of M13 Phage Displaying Single Chain Antibody

HIV-1 Tat protein (McKesson BioServices) was coupled to a preactivatedsubstrate. After blocking with ethanolamine, the array was washed with0.005% Tween20 in phosphate buffered saline, pH 7, and then 0.1% BSA inphosphate buffered saline, pH 7. A serial dilution of M13 phagedisplaying single chain antibody against the Tat protein was incubatedwith the Tat protein adsorbent array at 4° C. overnight. A negativecontrol of a serial dilution of M13 phage not displaying the singlechain antibody against the Tat protein was also incubated with the Tatprotein adsorbent array the same way. The arrays were washed with 0.05%Tween20 in phosphate buffered saline, followed by 1 M urea in phosphatebuffered saline, pH 7.0 and finally with water to remove buffer andurea. An aliquot of 0.3 μl of CHCA in 50% acetonitrile 0.5%trifluoroacetic acid was added. The retained phage was analyzed by laserdesorption/ionization time-of-flight mass spectrometer.

A specific binding of M13 phage displaying single chain antibody againstTat protein was observed in a concentration dependent manner (solidline). FIGS. 26A-26D. Nonspecific binding by a nonspecific M13 phage wasminimal on the adsorbent array (dashed line). These results illustrate avery sensitive method of detecting a phage containing a gene thatencodes a single chain antibody specifically recognizing a targetanalyte.

XI. SCREENING TO DETERMINE WHETHER A COMPOUND INHIBITS BINDING BETWEENRECEPTOR AND LIGAND

The methods of this invention can be used to determine whether a testagent modulates the binding of a ligand for a receptor. In this example,we show that retentate chromatography can be detect the inhibition ofbinding between TGF-β and bound TGF-β receptor used as an absorbent byfree TGF-β receptor.

TGF-β recombinant receptor-Fc fusion protein (R&D, Minnesota) wasspecifically bound on a Protein G adsorbent array. TGF-β (R&D,Minnesota) was serially diluted into cell conditioned medium(2.5×concentrated) and incubated with the receptor-Fc Protein Gadsorbent array at 4° C. overnight. Another set of serially dilutedTGF-β in cell conditioned medium was incubated with the receptor-FcProtein G adsorbent array in the presence of a modulating agent. In thisillustration, the modulating agent was the free TGF-β receptor. Afterincubation under the same conditions, the chips were washed with 0.05%Triton X100 in PBS and then 3M urea in PBS. An aliquot of 0.3 μl ofsinapinic acid was added to each spot and analyzed by laserdesorption/ionization time-of-flight mass spectrometry.

FIG. 27A shows the specific binding of 1 μg/ml TGF-β to the receptor-FcProtein G adsorbent array (solid line). Little or no proteins in thecell conditioned medium were found to bind. FIG. 27B shows the specificbinding of 100 ng/ml of TGF-β to the receptor-Fc Protein G adsorbentarray (solid line). When the incubation of TGF-β and the receptor-FcProtein G adsorbent array was performed in the presence of a modulatingagent (free TGF-β receptor), the binding was completely eliminated whenthere was 100 ng/ml of TGF-β (FIG. 27A, dashed line) and only a trace ofbinding where there was 1 μg/ml of TGF-β (FIG. 27B, dashed line). Inthis illustration, the modulating agent (the same receptor) was highspecific binding firmly affinity for the ligand, thus offering a veryeffective competition of the target analyte binding event. In the othercases, the ratio of the target analyte bound to the adsorbent in thepresent and absence of the modulating agent gives an indication of theefficacy of the modulating agent.

XII. RESOLVING POWER OR RETENTATE CHROMATOGRAPHY

This example demonstrates the ability of retentate chromatography, withits parallel processing of a sample under different selectivityconditions, to resolve proteins in a sample.

Hemophilus influenzae lysate was prepared in 10% glycerol. Aftercentrifugation, the supernate was diluted 1:3 in 0.01% Triton X100 in 25mM HEPES, pH 7.4. An aliquot of 2 μl of the diluted sample was added toa spot of adsorbent array cationic site. After incubation at roomtemperature for 30 min, the spot was washed with 25 mM HEPES, pH 7.4. Asecond aliquot of 2 μl of the dilute sample as added to a spot ofadsorbent array aliphatic hydrophobic site. After incubation at roomtemperature for 30 min, the spot was washed with water. A third aliquotof 2 μl of the diluted sample was added to a spot of adsorbent arrayCu(II) site. After incubation at room temperature for 30 min, the spotwas washed with 0.05% Triton X100 in phosphate buffered saline, pH 7.4.An aliquot of 0.3 of sinapinic acid solution in 50% acetonitrile 0.5%trifluoroacetic acid was added to each spot. The retained analytes oneach site was analyzed with laser desorption/ionization time-of-flightmass spectrometer.

Results are shown in FIGS. 28-31. The total retained analyte count wasaround 550. The result illustrates a method for combinatorialseparation, including separation and detection of multiple analytes inparallel.

XIII. SEQUENTIAL ASSEMBLY OF MULTIMERIC STRUCTURES

This examples illustrates a method of building a secondary adsorbent ona primary adsorbent. The secondary adsorbent then acts as a specificadsorbent for a target analyte.

An aliquot of 0.5 μl of GST fusion receptor diluted in 20 mM Tris 100mM, sodium chloride. 0.4% NP40, pH 7.2, was added to a spot of anadsorbent array normal site. The solution was allowed to concentrate onthe spot until almost dryness. The spot was washed with 2 μl of 10 mMTris, 50 mM sodium chloride, pH 7.2, three times. Each wash wasaccomplished by pipeting the wash solution in and out of the spot fivetimes. The spot was finally washed with 2 μl of water two times toremove remaining buffer. An aliquot of 0.3 μl of sinapinic acid solutionin 50% acetonitrile, 0.5% trifluoroacetic acid was added to the spot.The retained GST fusion receptor was analyzed with laserdesorption/ionization time-of-flight mass spectrometer. (FIG. 32.)

An aliquot of 0.5 μl of GST fusion receptor in 20 mM Tris, 100 mM sodiumchloride, 0.4% NP40, pH 7.2, was added to a spot of an adsorbent arraynormal site. A sample containing only GST protein (with no receptor) wasapplied to another spot as a negative control. The solution was allowedto concentrate on the spot until almost dryness. 0.5 μl of 10 mM Tris,50 mM sodium chloride, pH 7.2, was added to each spot. The solution wasremoved using a pipet after 10 seconds of standing at room temperature.

An aliquot of 1 μl of a solution containing one specific ligand in alibrary of 96 other ligands was immediately added to each spot. Theadsorbent array was incubated in a moist chamber at room temperature for1 hour. Each spot was washed with 2 μl of 30% isopropanol:acetonitrile(1:2) in water, two times. Each wash was accomplished by pipeting thewash solution in and out of the spot ten times. An aliquot of 0.3 μl ofα-cyano-4-hydroxycinnamic acid solution in 50% acetonitrile, 0.5%trifluoroacetic acid was added to the spot. The captured ligand on thereceptor was analyzed with laser desorption/ionization time-of-flightmass spectrometer.

FIG. 33A shows the binding of a specific ligand out of a library of 96other ligands to the GST fusion receptor which is captured on anadsorbent array normal site. FIG. 33B shows that there is no binding ofthe ligand to GST protein alone (with no receptor) captured on the samearray, which serves as a negative control of the experiment.

The present invention provides novel materials and methods for retentatechromatography. While specific examples have been provided, the abovedescription is illustrative and not restrictive. Many variations of theinvention will become apparent to those skilled in the art upon reviewof this specification. The scope of the invention should, therefore, bedetermined not with reference to the above description, but insteadshould be determined with reference to the appended claims along withtheir full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument Applicants do not admit that any particular reference is “priorart” to their invention.

What is claimed is:
 1. A method for determining whether an agentmodulates binding between a receptor/ligand pair that specifically bindto each other, the method comprising the steps of: a) providing a massspectrometry probe, the probe comprising a substrate having a surfaceand a receptor or ligand of said receptor/ligand pair docked to asurface of the substrate through an adsorbent; b) exposing the receptoror the ligand docked to the substrate to its binding partner and to saidagent under an elution conditions that allows for binding between thereceptor and the ligand; c) removing unbound binding partner from thesurface of the substrate; d) measuring the amount of binding partnerretained on the docked receptor or ligand in the presence and absence ofthe agent by laser desorption mass spectrometry of any retained bindingpartner from the surface of the substrate; and e) determining whetherthe agent modulates binding by comparing the measured amount of bindingbetween the receptor and the ligand in the presence and absence of theagent whereby a difference between the measured amount of bindingbetween the receptor and ligand in the presence and absence of the agentindicates that the agent modulates binding between the receptor/ligandpair.
 2. The method of claim 1 wherein the receptor is bound to thesubstrate.
 3. The method of claim 1 wherein the ligand is bound to thesubstrate.
 4. The method of claim 1 wherein the receptor is a cellsurface receptor.
 5. The method of claim 1 wherein the receptor is anintracellular receptor.
 6. The method of claim 1 wherein the receptor isa hormone receptor.
 7. The method of claim 2 wherein the receptor iscomprised within a cell membrane.
 8. The method of claim 1 wherein thereceptor or the ligand docked to the substrate is docked to thesubstrate through a linker.
 9. The method of claim 1 wherein the agentis a small organic molecule having a size up to about 5000 daltons. 10.The method of claim 8, wherein the linker is a bifunctional linker. 11.The method of claim 1 wherein said agent inhibits binding between saidreceptor/ligand pair.
 12. The method of claim 1 further comprisingapplying a matrix material to the surface before laser desorption massspectrometry.
 13. The method of claim 1 wherein the probe furthercomprises energy absorbing molecules chemically bound to the surfacebefore exposing.